Inhibitors of glucose-6-phosphate dehydrogenase for treating cardiovascular and pulmonary conditions

ABSTRACT

The present disclosure provides for methods of treating or preventing a cardiovascular disorder and/or a related pulmonary disorder in a subject. In certain embodiments, the method comprises administering a therapeutically effective amount of an inhibitor of Glucose-6-phosphate dehydrogenase (G6PD), or a pharmaceutically acceptable salt, non-salt amorphous form, solvate, poly-morph, tautomer or prodrug thereof.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Divisional of U.S. application Ser. No.16/349,676, filed May 4, 2019, which is a U.S. National PhaseApplication under 35 U.S.C. § 371 of International Patent ApplicationNo. PCT/US2017/061741, filed Nov. 15, 2017, and claims the benefit ofpriority under 35 U.S.C. Section 119(e) of U.S. Application No.62/422,929, filed Nov. 16, 2016, all of which are incorporated byreference in their entireties. The International Application waspublished on May 24, 2018 as International Publication No. WO2018/093856 A1.

FIELD OF THE INVENTION

The present invention relates to treating or preventing cardiovasculardiseases using an inhibitor of glucose-6-phosphate dehydrogenase (G6PD).

BACKGROUND

Cardiovascular diseases are among the leading causes of mortality andmorbidity worldwide with ever-increasing prevalence. Cardiovasculardiseases include numerous conditions that affect the heart, heartvalves, blood, and blood vessels (arteries, capillaries, and veins) ofthe body. The causes of cardiovascular disease are diverse butatherosclerosis and/or hypertension are the most common. Risk factorsinclude elevated plasma total or LDL cholesterol, elevatedtriglycerides, low HDL cholesterol, e.g. hyperlipidemia,hypercholesterolemia, or hypoalphalipoproteinemia, and increasedinflammatory markers such as C-reactive protein and fibrinogen.

Major cardiovascular diseases including stroke, atherosclerosis, andhypertension, as well as orphan diseases such as pulmonary hypertension,angiosarcoma, hemangiosarcoma, and hypertrophic cardiomyopathies, areincurable. In addition, medical therapies to treat congestive heartfailure and pulmonary hypertension-associated heart failure areinadequate.

Pulmonary hypertension presents an increase of blood pressure in thepulmonary artery, pulmonary vein, or pulmonary capillaries, togetherknown as the lung vasculature, leading to shortness of breath,dizziness, fainting, leg swelling and other symptoms. Pulmonarycirculation is a low resistance, low pressure, and high compliantvascular bed. In pulmonary hypertension, the pressure in the pulmonaryartery rises above normal levels. Normally, pulmonary artery pressure ismaintained around 20-25 mmHg Pulmonary hypertension is defined when thepressures increase to more than 30 mmHg Pulmonary hypertension is amajor cause of morbidity and mortality in patients with severaldifferent clinical conditions. Pulmonary hypertension is a progressivedisease and the pathophysiology of pulmonary hypertension isheterogeneous. Severe pulmonary hypertension remains debilitating anddeadly. Pulmonary hypertension is divided into five groups with diverseetiologies. In all forms of pulmonary hypertension, pulmonary arterypressure increases mainly because of increased pulmonaryconstriction/resistance and narrowing or remodeling of pulmonary arteryand veins. One cause of pulmonary hypertension is alveolar hypoxia,which results from localized inadequate ventilation of well-perfusedalveoli or from a generalized decrease in alveolar ventilation.Pulmonary hypertension is also a vascular permeability related disease.Current therapies are inadequate to reverse the complex pulmonaryvascular remodeling and reduce pulmonary vascular resistance. Pulmonaryhypertension has been historically chronic and incurable with a poorsurvival rate. Treatment of pulmonary hypertension usually involvescontinuous use of oxygen. Pulmonary vasodilators (e.g., hydralazine,calcium blockers, nitrous oxide, prostacyclin) have not proveneffective, and lung transplant is often required for patients who do notrespond to therapy.

Arteriosclerosis, which is induced and progressed by various riskfactors, causes thickening of the arterial lumen to interrupt bloodflow, resulting in a cardiovascular disease such as aortic aneurysm,angina, myocardial infarction, or cerebral infarction.

Cardiac hypertrophy is an adaptive response of the heart cells toelevated levels of biomechanical stress imposed by a variety ofextrinsic and intrinsic stimuli including pressure or volume overload,familial/genetic cardiomyopathies, or loss of contractile mass frompreceding infarction (Frey et al. (2004) Circulation 109:1580-1589; Freyet al. (2003) Annu. Rev. Physiol. 65:45-79; Yoshida et al. (1986) J.Cardiogr. 16:399-406). If sustained, hypertrophy often becomespathological, accompanied by significant risk of arrhythmia, progressionto heart failure, and sudden death (Frey et al. (2004), supra; Levy etal. (1990) N. Engl. J. Med. 322:1561-1566; Koren et al. (1991) Ann.Intern. Med. 114:345-352). At the molecular level, pathologicalhypertrophy is associated with re-induction of the so-called fetal geneprogram in which the fetal isoforms of genes responsible for regulatingcardiac contractility and calcium handling (e.g. .beta.-MHC) areupregulated (Frey et al. (2004), supra; Frey et al. (2003), supra);Olson (2004) Nat. Med. 10:467-474; Iemitsu et al. (2001) Am. J. Physiol.Regul. Integr. Comp. Physiol. 281:R2029-2036). At the cellular level,the main characteristics of ventricular hypertrophic growth are enhancedprotein synthesis and an increase in size of cardiomyocytes (Frey et al.(2004), supra; Frey et al. (2003), supra). As pathologic hypertrophyprogresses, these changes in molecular and cellular phenotypes areaccompanied by an increase in apoptosis, fibrosis, chamber dilation, anddecreased systolic function (Frey et al. (2004), supra).

Heart failure is associated with high morbidity as well as significantmortality. The clinical syndrome of heart failure is the result ofheterogeneous myocardial or vascular diseases, and is defined byinsufficiency to maintain blood circulation throughout the body. Despitesignificant advances in the clinical management of heart failure,conventional therapies are ultimately ineffective in many patients whoprogress to advanced heart failure. In these cases, implantation of leftventricular assist devices (LVAD) and/or heart transplantation can bethe only viable options.

In view of the foregoing, there is a need to develop effectivetreatments for various cardiovascular disorders. In this disclosure,novel therapies to treat cardiovascular disorders, such as pulmonaryhypertension, pulmonary hypertension-associated heart failure, atrialfibrillation or arrhythmia, and cardiomyopathies are described.

SUMMARY OF THE INVENTION

In certain embodiments, the present invention relates to a method fortreating or preventing a cardiovascular disorder and/or a pulmonarydisorder in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of an inhibitor ofGlucose-6-phosphate dehydrogenase (G6PD), or a pharmaceuticallyacceptable salt, solvate, poly-morph, tautomer or prodrug thereof.

In certain embodiments, the inhibitor comprises a compound having theformula of Formula I, Formula II, Formula III, Formula IV, Formula V,Formula IV, Formula VII, or Formula VIII, or any combination thereof.

In additional embodiments, the inhibitor comprises N-[(3β,5α)-20-Oxopregnan-3-yl] methanesulfonamide;N-ethyl-N′-[(3β,5α)-17-oxoandrostan-3-yl]urea;(3β,5α)-3,21-Dihydroxypregnan-20-one, or any combination thereof.

In additional embodiments, the cardiovascular disorder comprisespulmonary hypertension, pulmonary hypertension-associated heart failure,congestive heart failure, cardiomyopathies, arrhythmia including atrialfibrillation, hypertension, stroke, medial hypertrophy, acute neointimalformation following iatrogenic interventions, atherosclerosis, orcombinations thereof.

In further embodiments, the cardiovascular disorder and/or pulmonarydisorder comprises angiosarcoma, hemangioscarcoma, Timothy Syndrome,hypertrophic cardiomyopathy, or combinations thereof. In additionalembodiments, the cardiovascular disorder and/or pulmonary disordercomprises any of the groups of pulmonary hypertension 1-5, orcombinations thereof. In additional embodiments, the disorder comprisesscleroderma, categorized as pulmonary hypertension group I.

In certain embodiments, the method further comprises treating thesubject with a diuretic, a vasodilator, an inotropic agent, anangiotensin converting enzyme (ACE) inhibitor, a beta blocker, aneurohumoral blocker, an aldosterone antagonist, histone deactylaseinhibitors, erythropoietin, or combinations thereof.

In certain embodiments, the method further comprises treating thesubject with a medical device and/or surgery. In certain embodiments,the medical device comprises a bi-ventricular pacemaker, an implantablecardioverter-defibrillator (ICD), a ventricular assist device (VAD), aleft ventricular assist device (LVAD), a cardiac resynchronizationtherapy (CRT), or combinations thereof.

In certain embodiments, the present invention relates to use of acompound having the formula of Formula I, Formula II, Formula III,Formula IV, Formula V, Formula IV, Formula VII, or Formula VIII, or anycombination thereof, or a pharmaceutically acceptable salt (crystaland/or amorphous), non-salt amorphous form, solvate, poly-morph,tautomer or prodrug thereof in the preparation of a pharmaceuticalcomposition for treating or preventing a cardiovascular disorder and/ora pulmonary disorder in a subject in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structures of Compound 7e, 14f and 34. Circles indicatehydrogen residue replacement with CH3 or O inactivates the drug frominhibiting G6PD and lose their cardiovascular actions.

FIG. 2 is a table showing results for mice exposed to hypoxia (10% O₂)or to ambient atmosphere (21% O₂) for 5 wk. These mice developedpulmonary hypertension (PH group). Mice were treated with PD2124(Compound 34) s.c. for 1 wk. Body weight and hematocrit was measured.

FIGS. 3A and 3B are graphs showing results for bovine pulmonary arteriesthat were incubated in Krebs buffer with and without G6PD inhibitorPD2958 (2958) and PD2124 (2124) for 12 hr. Vascular smooth cell-specificprotein myocardin (MYOCD; FIG. 3A) and smooth muscle myosin heavy chain(MHY11; FIG. 3B) expression was increased by PD2958 (10 microM) andPD2124 (1 microM).

FIGS. 4A and 4B are graphs showing results for rat pulmonary arterysmooth muscle cells that were cultured in 15% DMEM media for 48 hr andcells were treated with newly synthesized G6PD drugs; PD109 (109),PD2124 (2124), PD2958 (2958) and PD4091 (4091) or vehicle control (Con)for 72 hr. Cell number/proliferation (FIG. 4A) and apoptosis (FIG. 4B)was determined by Cyquant and Caspase3/7 assay, respectively. Cellnumbers were decreased by G6PD inhibitors (FIG. 4A). Cell apoptosis wasincreased by PD2958 (1 microM) N=5 in each group (FIG. 4B).

FIGS. 5A and 5B are images (FIG. 5A) and a graph (FIG. 5B) showing ratpulmonary artery smooth muscle cells that were cultured in 15% DMEMmedia for 48 hr and cells were treated with newly synthesized G6PDdrugs; PD2958 (2958) or vehicle control (Con) for 72 hr. Cells migrationwas determined. Cell migration was abrogated by PD2958 (1 microM). N=5in each group.

FIG. 6 is a graph showing bovine pulmonary artery rings that wereincubated in Krebs buffer and pre-contracted with KCl (30 mM). Thesepre-contracted rings relaxed by G6PD inhibitors; PD109 (109), PD2124(2124), PD2958 (2958) and PD4091 (4091) or dehydroepiandrosterone(DHEA).

FIG. 7 is a graph showing aortic rings of wild-type and G6PD-deficientpre-contracted with KCl (30 mM). These rings from wild-type but notG6PD-deficient mice were relaxed in dose-dependent manner by PD2958(2958).

FIGS. 8A-8C are graphs showing results for mice that were divided intofour groups and treated under the following conditions: control (ctrl)exposed to ambient atmosphere (21% O₂); pulmonary hypertension exposedto 10% O₂ (PH); PH treated with inactive G6PD inhibitor (PH_Placebo);and PH treated with active G6PD inhibitor (PH_2124) for 5 wk. Placeboand 2124 were injected S.C. (1.3 mg/Kg) for 1 wk from wk 4 to wk 5. G6PDinhibitor 2124 reduced and reversed pulmonary resistance determined asPAAT-to-ET ratio (FIG. 8A), arterial elastance (FIG. 8B), and TPR (FIG.8C).

FIGS. 9A and 9B are graphs showing results for mice that were divided infour groups and treated under the following conditions: control (ctrl)exposed to ambient atmosphere (21% O₂); pulmonary hypertension exposedto 10% O₂ (PH); PH treated with inactive G6PD inhibitor (PH_Placebo);and PH treated with active G6PD inhibitor (PH_2124) for 5 wk. Placeboand 2124 were injected S.C. (1.3 mg/Kg) for 1 wk from wk 4 to wk 5. G6PDinhibitor 2124 reduced and reversed left ventricle (LV) stiffness (FIG.9A) and increased cardiac index (FIG. 9B).

DETAILED DESCRIPTION

As described herein we provide methods as well as one or moreagents/compounds that inhibit G6PD for the treatment, prophylaxis oralleviation of cardiovascular conditions described herein, or relatedpulmonary conditions, or predisposition to such a condition.

The present disclosure provides for methods of treating or preventing acardiovascular disorder and/or a related pulmonary disorder in asubject. In the method, a therapeutically effective amount of aninhibitor of Glucose-6-phosphate dehydrogenase (G6PD) is administered.The inhibitor may be a compound having the formula according to FormulaI, Formula II, Formula III, Formula IV, Formula V, Formula IV, FormulaVII, or Formula VIII.

In certain embodiments, the present composition comprises one or more ofthe following compounds: N-[(3β, 5α)-20-Oxopregnan-3-yl]methanesulfonamide; N-ethyl-N-[(3β, 5α)-17-oxoandrostan-3-yl]urea; and(3β,5α)-3,21-Dihydroxypregnan-20-one. Table 1 lists the structures ofexemplary compounds of the present disclosure.

TABLE 1 Compound Chemical Name Structure Synthesis PD109 (109)5-Androsten-3- beta-hydroxy- 17-one (Dehydro- epiandrosterone)

(Commercially available, CAS Number 53-43-0) PD2124 (2124); Compound 34(3β,5α)-3,21- Dihydroxy- pregnan-20-one

Example 2 PD2958 (2958); Compound 7e N-Ethyl-N′- [(3β,5α)-17-oxo-androstan-3-yl]- urea

Example 2 PD4091 (4091); Compound 14f N-[(3β,5α)-20- Oxopregnan-3-yl]methanesul- fonamide

Example 2

In yet additional embodiments, the invention relates to a pharmaceuticalcomposition comprising a pharmaceutically effective amount of a compoundof Formula I, Formula II, Formula III, Formula IV, Formula V, FormulaIV, Formula VII, or Formula VIII, or a pharmaceutically acceptable salt(crystal and/or amorphous), non-salt amorphous form, solvate,poly-morph, tautomer or prodrug thereof. In certain embodiments, thecompound further comprises a pharmaceutically acceptable carrier,diluent, excipient and/or adjuvant.

In certain embodiments, the present agent/composition is specificallyadministered to lung or cardiac cells, to inhibit gene function andprevent one or more of the symptoms and processes associated with theprogression of cardiovascular or pulmonary conditions. Such treatmentmay also be useful in treating patients who already exhibitcardiovascular or pulmonary conditions, to reverse or alleviate one ormore of the disease processes. Additionally, approaches utilizing one ormore additional inhibitors including an inhibitor of protein kinase Gand blocker of endothelin A or B receptor or any combination of these,are also expected to be useful for treating certain conditions.

In an embodiment, “patient” or “subject” refers to mammals and includeshuman and veterinary subjects, including avians. In an embodiment, thesubject is mammalian.

Glucose-6-Phosphate Dehydrogenase (G6PD)

Glucose-6-phosphate dehydrogenase (G6PD) generates nicotinamide adeninedinucleotide phosphate reduced (NADPH), a key cofactor for variousredox-sensitive enzymes like: NADPH oxidases, glutathione/thioredoxinreductases, and other reductive and anabolic reactions in the cell(Gupte and Wolin (2012): Hypertension; 60:269-275). We have shown thatG6PD is involved in regulation of coronary and pulmonary arterycontraction and relaxation (Ata et al (2011): Am J Physiol Heart CircPhysiol; 300:H2054-H2063), and pulmonary artery SMC phenotype(Chettimada et al (2012): Am J Physiol Lung Cell Mol Physiol;303:L64-L74). Glucose-6-phosphate dehydrogenase has been shown to beassociated with progressive pulmonary artery remodeling in pulmonaryhypertension.

Glycolysis, glucose flux through the PPP, and the activity of NADPHproducing isocitrate dehydrogenase-1 and -2 are increased in pulmonaryartery of idiopathic- and heritable-pulmonary hypertension patients, andin endothelial cells and fibroblasts from idiopathic pulmonaryhypertension patients. G6PD expression and activity are increased in:(a) endothelin-1 treated pulmonary artery smooth muscle cells frompulmonary hypertension patients; (b) hypoxic cultured rat pulmonaryartery smooth muscle cells; and (c) lungs of pulmonary hypertensive ratmodels. G6PD is a major supplier of NADPH (60% by G6PD+40% by isocitratedehydrogenase) for: anabolic reactions and superoxide production fromNADPH oxidases in the cell. Excess NADPH generation contributes topathogenic “reductive stress” in cardiovascular system.

G6PD-derived NADPH plays a key role in stimulating proliferation andinhibiting apoptosis of cells (Buchakjian and Kornbluth (2010): Nat RevMol Cell Biol; 11:715-727). Ectopic expression of G6PD increases ratPASMC proliferation (Chettimada et al (2015): Am J Physiol Lung Cell MolPhysiol; 308:L287-L300) and contributes to the HIFI α-inducedendothelial growth (Leopold et al (2003): J Biol Chem; 278:32100-32106).Additionally, our findings suggest that hyper-activation of G6PD inCD133k progenitor cells promote their self-renewal (Chettimada et al(2014): Am J Physiol Lung Cell Mol Physiol; 307:L545-L556). CD133⁺ cellspotentially participate in the PA remodeling process in PAH (Asosingh etal (2008): Am J Pathol; 172:615-627). Conversely, inhibition of G6PDincreases the rate of apoptosis of X laevis oocytes, HEK293 cells,esophageal squamous cell carcinoma, and melanoma cells (Bouchier-Hayeset al (2009): Mol Cell; 35:830-840; Nutt et al (2005): Cell; 35:89-103;Wang et al (2016): Tumout Biol; 37:781-789; Cai et al (2015): Am JCancer Res; 5:1610-1620). In PH, the PASMC and endothelial cellproliferation is accompanied by decreased expression of pro-apoptoticgenes (Bull et al (2007): Proc Am Thorac Soc; 4:117-120). Therefore,altogether these findings allude stimulation of G6PD activity byendothelin-1 or by hypoxia likely inhibits apoptosis and promotesproliferation of PASMC, and contributes to progressive PA remodeling andto the pathogenesis of HPH and PAH.

G6PD deficiency is common in humans, and several point mutations havebeen found in this enzyme in different ethnic groups around the world.Epidemiological studies suggest that individuals who harbor aMediterranean-type non-synonymous mutation [single nucleotidepolymorphism in exon 6: dbSNP rs5030868] have 80% less G6PD activity ascompared to normal individuals and are less likely to havecardiovascular diseases (Gupte (2008): Curr Opin Investig Drugs9:993-1000), including sickle cell anemia-associated PH.

Illustrative nucleotide sequences encoding the amino acid sequences ofhuman G6PD are known and published, e.g., in GenBank Accession Nos.NM_000402, AH003054.2 etc.

In some embodiments, the level of G6PD is decreased in a cardiac and/orlung cells. In certain embodiments, treatment may be targeted to, orspecific to, diseased cells. The expression of G6PD may be specificallydecreased only in diseased cells (i.e., those cells which arepredisposed to the cardiovascular condition and/or related pulmonarycondition, or exhibiting cardiovascular condition and/or relatedpulmonary condition already), and not substantially in othernon-diseased cells. In these methods, expression of G6PD may not besubstantially reduced in other cells, i.e., cells which are non-diseasedcells. Thus, in such embodiments, the level of G6PD remainssubstantially the same or similar in non-diseased cells in the course ofor following treatment.

Cell specific reduction of G6PD levels and/or activity may be achievedby targeted administration, i.e., applying the treatment only to thetargeted cells and not other cells. However, in other embodiments,down-regulation of G6PD expression in other cells (e.g., a portion ofnon-diseased cells, and not substantially in other cell or tissue types)is employed.

The methods and compositions described here may reduce the level and/oractivity of G6PD, G6PD polynucleotides, G6PD nucleotides and G6PDnucleic acids, as well as variants, homologues, derivatives andfragments of any of these. The inhibitors targeting G6PD may also beused for the methods of treatment or prophylaxis described.

The terms “G6PD polynucleotide”, “G6PD nucleotide” and “G6PD nucleicacid,” “G6PD nucleic acid” may be used interchangeably, and should beunderstood to specifically include both cDNA and genomic G6PD sequences.These terms are also intended to include a nucleic acid sequence capableof encoding a G6PD polypeptide and/or a fragment, derivative, homologueor variant of this.

By “down-regulation” or “reduction” is meant any negative effect on thecondition being studied; this may be total or partial. Thus, where thelevel or activity of a protein is being detected, the present agent iscapable of reducing, ameliorating, or abolishing the level or activityof the protein. The down-regulation of the level or activity of theprotein achieved by the present agent may be at least 10%, such as atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90% or more compared to the level oractivity of the protein in the absence of the present agent.

The term “compound” refers to a chemical compound (naturally occurringor synthesized), such as a biological macromolecule (e.g., nucleic acid,protein, non-peptide, or organic molecule), or an extract made frombiological materials such as bacteria, plants, fungi, or animal(particularly mammalian) cells or tissues, or even an inorganic elementor molecule. The compound may be an antibody.

In some embodiments, the anti-G6PD agent is provided as an injectable orintravenenous composition and administered accordingly. The dosage ofthe anti-G6PD agent inhibitor may be between about 5 mg/kg/2 weeks toabout 10 mg/kg/2 weeks. The anti-G6PD agent inhibitor may be provided ina dosage of between 10-300 mg/day, such as at least 30 mg/day, less than200 mg/day or between 30 mg/day to 200 mg/day.

Inhibitors of G6PD

The present disclosure provides methods of treating or preventing acardiovascular disorder and/or a related pulmonary disorder in a subjectby administering one or more compounds as described herein. In oneembodiment, an androstane-derivative is used in the present method. Inanother embodiment, a preganane-derivative is used in the presentmethod. In certain embodiments, the steroid inhibition of G6PD has beensubstantially developed; the 3β-alcohol can be replaced with 3β-H-bonddonors such as sulfamide, sulfonamide, urea, and carbamate. In certainembodiments, improved potency was achieved by replacing the androstanenucleus with a pregnane nucleus, provided a ketone at C-20 is present.In certain embodiments, for pregnan-20-ones a 21-hydroxyl group isincorporated. J. Medicinal Chemistry 2012; 55:4431-4445.

In certain embodiments, the present compound has an IC50 in inhibitingG6PD activity ranging from about 0.1 μM to about 500 μM, from about 0.5μM to about 200 μM, from about 1 μM to about 100 μM, from about 1 μM toabout 50 μM, from about 1 μM to about 30 μM, or from about 1 μM to about10 μM. In certain embodiments, the present compound displays at least 10fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90fold, at least 100 fold, at least 150 fold, at least 180 fold, or atleast 200 fold, efficacy to inhibit G6PD activity in vitro and in vivothan dehydroepiandrosterone or epiandrosterone.

In accordance with the present invention, there is provided compounds ofFormula I or Formula II, and methods for treating a cardiovasculardisorder and/or a related pulmonary disorder in a patient, comprisingadministering to the patient any one of the compounds having Formula Ior Formula II:

In Formula I, R is urea, carbamate, sulfonamide or sulfamide.

In Formula II, R is sulfonamide or sulfamide; R′ is H or OH.

In accordance with the present invention, there is provided compounds ofFormula III or Formula IV, or V and methods for treating acardiovascular disorder and/or a related pulmonary disorder in apatient, comprising administering to the patient any one of thecompounds having Formula III, Formula IV or Formula V:

Wherein: R is R¹NH and OH;

R¹ is selected from H and moieties, NHacyl, CONHR², CONR³R⁴, CO₂R²,SO₂R², SO₂R⁵ andSO₂NH₂;R² is straight and branched alkyl of 1 to 6 carbon atoms;R³ and R⁴ are independently selected from H and straight and branchedalkyl of 1 to 6 carbon atoms;R⁵ is phenyl optionally substituted with 1 or 2 substituents selectedfrom fluorine, chlorine, and straight and branched branched alkyl of 1to 6 carbon atoms.

The compounds of Class/Formula III may be obtained as inorganic ororganic pharmaceutically acceptable salts using methods known to thoseskilled in the art (Richard C. Larock, Comprehensive OrganicTransformations, VCH Publishers, 411-415, 1989). It is well known to oneskilled in the art that an appropriate salt which include but notlimited to inorganic salts which may be sodium, calcium, potassium ormagnesium and the like or equivalent thereof or hydrochloric,hydrobromic, hydroiodic, phosphoric, nitric or sulfate and the like.

Where:

R¹ is selected from H and moieties, NHacyl, CONHR², CONR³R⁴, CO₂R²,SO₂R², SO₂R⁵ andSO₂NH₂;R² is straight and branched alkyl of 1 to 6 carbon atoms;R³ and R⁴ are independently selected from H and straight and branchedalkyl of 1 to 6 carbon atoms;

R⁵ is phenyl optionally substituted with 1 or 2 substituents selectedfrom fluorine, chlorine, and straight and branched alkyl of 1 to 6carbon atoms. The compounds of Class/Formula IV may be obtained asinorganic or organic pharmaceutically acceptable salts using methodsknown to those skilled in the art (Richard C. Larock, ComprehensiveOrganic Transformations, VCH Publishers, 411-415, 1989). It is wellknown to one skilled in the art that an appropriate salt which includebut not limited to inorganic salts which may be sodium, calcium,potassium or magnesium and the like or equivalent thereof orhydrochloric, hydrobromic, hydroiodic, phosphoric, nitric or sulfate andthe like.

Wherein: R is R¹NH and OH;

R¹ is selected from H and moieties, NHacyl, CONHR², CONR³R⁴, CO₂R²,SO₂R², SO₂R⁵ andSO₂NH₂;R² is straight and branched alkyl of 1 to 6 carbon atoms;R³ and R⁴ are independently selected from H and straight and branchedalkyl of 1 to 6 carbon atoms;R⁵ is phenyl optionally substituted with 1 or 2 substituents selectedfrom fluorine, chlorine, and straight and branched alkyl of 1 to 6carbon atoms;

R⁶ is H or OH.

The compounds of Class/Formula V may be obtained as inorganic or organicpharmaceutically acceptable salts using methods known to those skilledin the art (Richard C. Larock, Comprehensive Organic Transformations,VCH Publishers, 411-415, 1989). It is well known to one skilled in theart that an appropriate salt which include but not limited to inorganicsalts which may be sodium, calcium, potassium or magnesium and the likeor equivalent thereof or hydrochloric, hydrobromic, hydroiodic,phosphoric, nitric or sulfate and the like.

In certain embodiments, the present composition comprises compounds ofFormula VI, Formula VII, or Formula VIII. In certain embodiments, thepresent composition comprises one or more of the following compounds:N-[(3β,5α)-20-Oxopregnan-3-yl] methanesulfonamide (Formula VI,R=sulfonamide, also called “compound 14f” herein); N-ethyl-N-[(3β,5α)-17-oxoandrostan-3-yl]urea (Formula VII, R=Urea, also called“compound 7e” herein); or (3β,5α)-3,21-Dihydroxypregnan-20-one (FormulaVIII, R═OH; R′═O; also called “compound 34” herein).

The compounds used in the methods of the present invention include allhydrates, solvates, and complexes of the compounds used by thisinvention. If a chiral center or another form of an isomeric center ispresent in a compound of the present invention, all forms of such isomeror isomers, including enantiomers and diastereomers, are intended to becovered herein. Compounds containing a chiral center may be used as aracemic mixture, an enantiomerically enriched mixture, or the racemicmixture may be separated using well-known techniques and an individualenantiomer may be used alone. The compounds described in the presentinvention are in racemic form or as individual enantiomers. Theenantiomers can be separated using known techniques, such as thosedescribed in Pure and Applied Chemistry 69, 1469-1474, (1997) IUPAC. Incases in which compounds have unsaturated carbon-carbon double bonds,both the cis (Z) and trans (E) isomers are within the scope of thisinvention. In cases wherein compounds may exist in tautomeric forms,such as keto-enol tautomers, each tautomeric form is contemplated asbeing included within this invention whether existing in equilibrium orpredominantly in one form.

When the structure of the compounds used in this invention includes anasymmetric carbon atom such compound can occur as racemates, racemicmixtures, and isolated single enantiomers. All such isomeric forms ofthese compounds are expressly included in this invention. Eachstereogenic carbon may be of the R or S configuration. It is to beunderstood accordingly that the isomers arising from such asymmetry(e.g., all enantiomers and diastereomers) are included within the scopeof this invention, unless indicated otherwise. Such isomers can beobtained in substantially pure form by classical separation techniquesand by stereochemically controlled synthesis, such as those described in“Enantiomers, Racemates and Resolutions” by J. Jacques, A. Collet and S.Wilen, Pub. John Wiley & Sons, N Y, 1981. For example, the resolutionmay be carried out by preparative chromatography on a chiral column.

The subject invention is also intended to include use of all isotopes ofatoms occurring on the compounds disclosed herein. Isotopes includethose atoms having the same atomic number but different mass numbers. Byway of general example and without limitation, isotopes of hydrogeninclude tritium and deuterium. Isotopes of carbon include carbon-13 andcarbon-14.

It will be noted that any notation of a carbon in structures throughoutthis application, when used without further notation, are intended torepresent all isotopes of carbon, such as ¹²C, ¹³C, or ¹⁴C. Furthermore,any compounds containing ¹³C or ¹⁴C may specifically have the structureof any of the compounds disclosed herein.

It will also be noted that any notation of a hydrogen in structuresthroughout this application, when used without further notation, areintended to represent all isotopes of hydrogen, such as ¹H, ²H, or ³H.Furthermore, any compounds containing ²H or ³H may specifically have thestructure of any of the compounds disclosed herein.

Isotopically-labeled compounds can generally be prepared by conventionaltechniques known to those skilled in the art or by processes analogousto those described in the Examples disclosed herein using appropriateisotopically-labeled reagents in place of the non-labeled reagentsemployed.

The compounds of the instant invention may be in a salt form. As usedherein, a “salt” is salt of the instant compounds which has beenmodified by making acid or base, salts of the compounds. In the case ofcompounds used for treatment of cancer, the salt is pharmaceuticallyacceptable. Examples of pharmaceutically acceptable salts include, butare not limited to, mineral or organic acid salts of basic residues suchas amines; alkali or organic salts of acidic residues such as phenols.The salts can be made using an organic or inorganic acid. Such acidsalts are chlorides, bromides, sulfates, nitrates/nitrites, esters,phosphates, sulfonates, formates, tartrates, maleates, malates,citrates, benzoates, salicylates, ascorbates, and the like. Phenolatesalts are the alkaline earth metal salts, sodium, potassium or lithium.The term “pharmaceutically acceptable salt” in this respect, refers tothe relatively non-toxic, inorganic and organic acid or base additionsalts of compounds of the present invention. These salts can be preparedin situ during the final isolation and purification of the compounds ofthe invention, or by separately treating a purified compound of theinvention in its free base or free acid form with a suitable organic orinorganic acid or base, and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, phosphate, nitrate/nitrite, ester, acetate, valerate, oleate,palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.(See, e.g., Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci.66:1-19).

As used herein, “alkyl” includes both branched and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms and may be unsubstituted or substituted. The Alkyls areC1-C10 alkyls, or a subset or individual thereof. In a non-limitingexample, where the alkyl is C1-C5 as in “C1-C5 alkyl”, it is defined toinclude groups having 1, 2, 3, 4 or 5 carbons in a linear or branchedarrangement and specifically includes methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, and pentyl. Alkyl may optionally besubstituted with phenyl or substituted phenyl to provide substituted orunsubstituted benzyl.

Heterocyclyl means a saturated or partially unsaturated monocyclicradical containing 3 to 8 ring atoms and preferably 5 to 6 ring atomsselected from carbon or nitrogen but not limited to pyrrolidine.

As used herein the term “aryl” refers to aromatic monocyclic ormulticyclic groups containing from 5 to 15 carbon atoms. Aryl groupsinclude, but are not limited to groups such as unsubstituted orsubstituted phenyl. When referring to said aryl being substituted, saidsubstitution may be at any position on the ring, other than the point ofattachment to the other ring system of a compound of the invention.Therefore, any hydrogen atom on the aryl ring may be substituted with asubstituent defined by the invention. In embodiments where the aryl is aphenyl ring, said substitution may be at the meta- and/or ortho- and/orpara-position relative to the point of attachment. Aryl may optionallybe substituted with a heterocyclyl-C(O)— moiety which includes apyrrolidinyl-C(O)— moiety.

The term “heteroaryl” as used herein, represents a stable monocyclic,bicyclic or polycyclic ring of up to 10 atoms in each ring, wherein atleast one ring is aromatic and contains from 1 to 4 heteroatoms orparticularly 1 to 2 heteroatoms selected from the group consisting of O,N and S. Bicyclic aromatic heteroaryl groups include phenyl, pyridine,pyrimidine or pyridazine rings that are (a) fused to a 6-memberedaromatic (unsaturated) heterocyclic ring having one nitrogen atom; (b)fused to a 5- or 6-membered aromatic (unsaturated) heterocyclic ringhaving two nitrogen atoms; (c) fused to a 5-membered aromatic(unsaturated) heterocyclic ring having one nitrogen atom together witheither one oxygen or one sulfur atom; or (d) fused to a 5-memberedaromatic (unsaturated) heterocyclic ring having one heteroatom. selectedfrom O, N or S. Heteroaryl groups within the scope of this definitioninclude but are not limited to: benzoimidazolyl, benzofuranyl,benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl,benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, indolinyl,indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl,isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl,oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl,pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl,pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrazolyl,tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl,azetidinyl, aziridinyl, 1,4-dioxanyl, hexahydroazepinyl,dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl,dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl,dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl,dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyriinidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, tetrahydrothienyl, acridinyl,carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl,benzotriazolyl, benzothiazolyl, benzoxazolyl, isoxazolyl, isothiazolyl,furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl,oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl,pyrimidinyl, pyrrolyl, tetra-hydroquinoline. In cases where theheteroaryl substituent is bicyclic and one ring is non-aromatic orcontains no heteroatoms, it is understood that attachment is via thearomatic ring or via the heteroatom containing ring, respectively. Ifthe heteroaryl contains nitrogen atoms, it is understood that thecorresponding N-oxides thereof are also encompassed by this definition.

In the compounds of the present invention, the alkyl, aryl, orheteroaryl groups can be further substituted by replacing one or morehydrogen atoms be alternative non-hydrogen groups. These include, butare not limited to, 1-4 groups selected from alkyl, alkoxy, halo,hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.

The term “substituted” refers to a functional group as described abovein which one or more bonds to a hydrogen atom contained therein arereplaced by a bond to non-hydrogen or non-carbon atoms, provided thatnormal valencies are maintained and that the substitution results in astable compound. Substituted groups also include groups in which one ormore bonds to a carbon (s) or hydrogen (s) atom are replaced by one ormore bonds, including double or triple bonds, to a heteroatom. Examplesof substituent groups include the functional groups described above,and, in particular, halogens (i.e., F, Cl, Br, and I); alkyl groups,such as methyl, ethyl, n-propyl, isopropryl, n-butyl, tert-butyl, andtrifluoromethyl; hydroxyl; alkoxy groups, such as methoxy, ethoxy,n-propoxy, and isopropoxy; aryloxy groups, such as phenoxy;arylalkyloxy, such as benzyloxy (phenylmethoxy) andp-trifluoromethylbenzyloxy (4-trifluoromethylphenylmethoxy);heteroaryloxy groups; sulfonyl groups, such as trifluoromethanesulfonyl,methanesulfonyl, and p-toluenesulfonyl; nitro, nitrosyl; mercapto;sulfanyl groups, such as methylsulfanyl, ethylsulfanyl andpropylsulfanyl; cyano; heterocyclyl-C(O)-moiety; amino groups, such asamino, methylamino, dimethylamino, ethylamino, and diethylamino; andcarboxyl. Where multiple substituent moieties are disclosed or claimed,the substituted compound can be independently substituted by one or moreof the disclosed or claimed substituent moieties, singly or plurally. Byindependently substituted, it is meant that the (two or more)substituents can be the same or different.

It is understood that substituents and substitution patterns on thecompounds of the instant invention can be selected by one of ordinaryskill in the art to provide compounds that are chemically stable andthat can be readily synthesized by techniques known in the art, as wellas those methods set forth below, from readily available startingmaterials. If a substituent is itself substituted with more than onegroup, it is understood that these multiple groups may be on the samecarbon or on different carbons, so long as a stable structure results.

In choosing the compounds of the present invention, one of ordinaryskill in the art will recognize that the various substituents, i.e. R₁,R₂, etc. are to be chosen in conformity with well-known principles ofchemical structure connectivity. Moreover, where hydrogens are not shownin the carbon-based structures herein, implicit hydrogens are understoodto complete valences as required.

The compounds of the instant invention may be in a salt form. As usedherein, a “salt” is salt of the instant compounds which has beenmodified by making acid or base, salts of the compounds. In the case ofcompounds used for treatment of cancer, the salt is pharmaceuticallyacceptable. Examples of pharmaceutically acceptable salts include, butare not limited to, mineral or organic acid salts of basic residues suchas amines; alkali or organic salts of acidic residues such as phenols.The salts can be made using an organic or inorganic acid. Such acidsalts are chlorides, bromides, sulfates, nitrates/nitrites, esters,phosphates, sulfonates, formates, tartrates, maleates, malates,citrates, benzoates, salicylates, ascorbates, and the like. Phenolatesalts are the alkaline earth metal salts, sodium, potassium or lithium.The term “pharmaceutically acceptable salt” in this respect, refers tothe relatively non-toxic, inorganic and organic acid or base additionsalts of compounds of the present invention. These salts can be preparedin situ during the final isolation and purification of the compounds ofthe invention, or by separately reacting a purified compound of theinvention in its free base or free acid form with a suitable organic orinorganic acid or base, and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, phosphate, nitrate/nitrite, ester, acetate, valerate, oleate,palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.(See, e.g., Berge et al. (1977) “Pharmaceutical Salts”, J″. Pharm. Sci.66:1-19).

Where a numerical range is provided herein for any parameter, it isunderstood that all numerical subsets of that numerical range, and allthe individual integer values contained therein, are provided as part ofthe invention. Thus, C1-C10 alkyl includes the subset of alkyls whichare 1-3 carbon atoms, the subset of alkyls which are 2-5 carbon atomsetc. as well as an alkyl which has 1 carbon atom, an alkyl which has 3carbon atoms, an alkyl which has 10 carbon atom, etc.

In an embodiment, the purines discussed herein are one or more ofadenosine, inosine, hypoxanthine, or adenine. In an embodiment,“determining” as used herein means experimentally determining.

The term “composition”, as in pharmaceutical composition, is intended toencompass a product comprising the active ingredient(s), and the inertingredient(s) (pharmaceutically acceptable excipients) that make up thecarrier, as well as any product which results, directly or indirectly,from combination, complexation or aggregation of any two or more of theingredients, or from dissociation of one or more of the ingredients, orfrom other types of reactions or interactions of one or more of theingredients. Accordingly, the pharmaceutical compositions of the presentinvention encompass any composition made by admixing a compound ofFormula I, Formula II, Formula III, Formula IV, Formula V, Formula IV,Formula VII, or Formula VIII, and pharmaceutically acceptableexcipients.

As used herein, the term “optionally” means that the subsequentlydescribed event(s) may or may not occur, and includes both event(s),which occur, and events that do not occur.

As used herein, the term “substituted with one or more groups” refers tosubstitution with the named substituent or substituents, multipledegrees of substitution, up to replacing all hydrogen atoms with thesame or different substituents, being allowed unless the number ofsubstituents is explicitly stated. Where the number of substituents isnot explicitly stated, one or more is intended.

As used herein, “a compound of the invention” means a compound ofFormula I, Formula II, Formula III, Formula IV, Formula V, Formula IV,Formula VII, or Formula VIII, or a salt, solvate or physiologicallyfunctional derivative thereof.

As used herein, the term “solvate” refers to a complex of variablestoichiometry formed by a solute (in this invention, a compound ofFormula I, Formula II, Formula III, Formula IV, Formula V, Formula IV,Formula VII, or Formula VIII, or a salt thereof) and a solvent. Suchsolvents for the purpose of the invention may not interfere with thebiological activity of the solute. Examples of suitable solventsinclude, but are not limited to, water, acetone, methanol, ethanol andacetic acid. Preferably the solvent used is a pharmaceuticallyacceptable solvent. Examples of suitable pharmaceutically acceptablesolvents include water, ethanol and acetic acid. Most preferably thesolvent is water.

As used herein, the term “physiologically functional derivative” refersto a compound (e.g, a drug precursor) that is transformed in vivo toyield a compound of Formula I, Formula II, Formula III, Formula IV,Formula V, Formula IV, Formula VII, or Formula VIII, or apharmaceutically acceptable salt, hydrate or solvate of the compound.The transformation may occur by various mechanisms (e.g., by metabolicor chemical processes), such as, for example, through hydrolysis inblood. Prodrugs are such derivatives, and a discussion of the use ofprodrugs is provided by T. Higuchi and W. Stella, “Pro-drugs as NovelDelivery Systems,” Vol. 14 of the A.C.S. Symposium Series, and inBioreversible Carriers in Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987.

All combinations of the various elements described herein are within thescope of the invention unless otherwise indicated herein or otherwiseclearly contradicted by context. Whilst the embodiments for eachvariable have generally been listed above separately for each variable,this invention also includes those compounds in which several or eachembodiment for compounds of Formula I, Formula II, Formula III, FormulaIV, Formula V, Formula IV, Formula VII, or Formula VIII, is selectedfrom each of the embodiments listed above. Therefore, this invention isintended to include all combinations of embodiments for each variable.

Any number of means for inhibiting G6PD activity or gene expression canbe used in the methods of the invention. It is noted that in addition toG6PD, G6PD isoforms or 6-phosphogluconorate dehydrogenase and otherenzymes in glucose (for example: malic enzyme or isocitratedehydrogenase) and fat metabolism (for example: malony-CoA and HMG-CoA)including cholesterol synthesis is another process which couldpotentially be blocked by inhibitory compounds in a similar manner asdescribed herein for G6PD.

Cardiovascular Diseases

The methods of the present invention may be used to treat a subjecthaving or at risk for cardiovascular disorders or cardiovasculardiseases.

Cardiovascular disorders or cardiovascular diseases can include anydisorders that affect the cardiovascular system, including the heartand/or blood vessels, such as arteries and veins. Cardiovasculardiseases can also include disorders affecting the kidneys. Non-limitingexamples of cardiovascular diseases include pulmonary hypertension,pulmonary hypertension-associated heart failure, hypertension, stroke,medial hypertrophy, acute neointimal formation following iatrogenicinterventions, atherosclerosis, congestive heart failure, heart failure,arrhythmia including atrial fibrillation, myocardial infarction,myocardial ischemia, cardiac hypertrophy, coronary heart disease,cardiac fibrosis, cardiomyopathy, ischemic heart disease, hypertensiveheart disease, inflammatory heart disease, valvular heart disease,diseases of the cardiac valves, atherosclerosis, cardiorenal disease,vascular damage, myocardial damage, cardiac valvular disease or othercardiac electrophysiologic abnormalities, hypertension, other cardiacdysfunction, and combinations thereof. Cardiovascular disease caninclude, but is not limited to, right-sided, left-sided failure orcongestive heart failure and could be due to any one of a number ofdifferent causes. Any type of cardiovascular disease, which includesimpaired functioning of either the left or right ventricle is alsoencompassed herein. In some embodiments, cardiovascular diseases includediabetes mellitus, hyperhomocysteinemia and hypercholesterolemia.

Non-limiting examples of cardiovascular diseases that may be treated bythe present composition and method also include angiosarcoma,hemangioscarcoma, Timothy Syndrome, hypertrophic cardiomyopathy, andcombinations thereof.

Pulmonary hypertension includes pulmonary hypertension groups 1-5,including scleroderma, which is a member of group 1 pulmonaryhypertension and is an autoimmune condition. (See: Ryan, John J. et al.“The WHO Classification of Pulmonary Hypertension: A Case-Based ImagingCompendium.” Pulmonary Circulation 2.1 (2012): 107-121. PMC. Web. 16Nov. 2016.)

Cardiomyopathies can include, but are not limited to, alcoholiccardiomyopathy, coronary artery disease, congenital heart disease,ischemic cardiomyopathy (ICM), dilated cardiomyopathy (DCM),hypertensive cardiomyopathy, valvular cardiomyopathy, inflammatorycardiomyopathy, diabetic cardiomyopathy and myocardiodystrophy, as wellas other forms of cardiomyopathies.

Hypertensive heart diseases can include, but are not limited to, leftventricular hypertrophy, coronary heart disease, heart failure(including congestive), hypertensive cardiomyopathy, cardiac arrhythmiasand renal disorders.

Inflammatory heart diseases can include, but are not limited to,endocarditis, inflammatory cardiomegaly and myocarditis.

Combination Therapy

The present composition may be administered alone or in combination witha second agent/treatment method (therapeutic intervention).

Therapeutic interventions that may be used in combination with thepresent composition or method can include, pharmacologic intervention,devices, surgical intervention, or any combination thereof.Pharmacologic interventions may include, but are not limited to,treatment with diuretics, vasodilators, inotropic agents (i.e.,compounds that increase cardiac contractility), ACE inhibitors,beta-blockers, neurohumoral blockers (e.g., beta-blockers, angiotensinconverting enzyme inhibitors), aldosterone antagonists (e.g.,spironolactone, eplerenone), histone deactylase inhibitors, anderythropoietin. Devices may include, e.g., a bi-ventricular pacemarker,implantable cardioverter-defibrillator (ICD), ventricular assist device(VAD), left ventricular assist device (LVAD), or cardiacresynchronization therapy (CRT). Surgical interventions may include,heart transplantation, artificial heart, etc.

In certain embodiments, therapeutic intervention can be implantation ofa medical device or surgical, which includes, for example, preventative,diagnostic or staging, curative and palliative surgery. Surgery may beused in conjunction with other therapies, including one or more otheragents as described herein. Such surgical therapeutic agents forvascular and cardiovascular diseases and disorders are well known tothose of skill in the art, and may include, but are not limited to,providing a cardiovascular mechanical prosthesis, angioplasty, coronaryartery reperfusion, catheter ablation, providing an implantablecardioverter defibrillator to the subject, mechanical circulatorysupport or a combination thereof. Examples of a mechanical circulatorysupport that may be used in the present invention comprise anintra-aortic balloon counterpulsation, left ventricular assist device(LVAD) or combinations thereof.

Pharmacologic agents for therapeutic interventions can include, but arenot limited to, miRNA based therapeutics (including antisenseoligonucleotides), antihyperlipoproteinemic agent, anantiarteriosclerotic agent, an antithrombotic/fibrinolytic agent, ablood coagulant, an antiarrhythmic agent, an antihypertensive agent, avasopressor, a treatment agent for congestive heart failure, anantianginal agent, an antibacterial agent or a combination thereof. U.S.Patent Application No. 2010/0317713.

An antihyperlipoproteinemic may be an agent that lowers theconcentration of one of more blood lipids and/or lipoproteins. Examplesof antihyperlipoproteinemics can include but are not limited to,acifran, azacosterol, benfluorex, p-benzalbutyramide, carnitine,chondroitin sulfate, clomestrone, detaxtran, dextran sulfate sodium, 5,8, 11, 14, 17-eicosapentaenoic acid, eritadenine, furazabol, meglutol,melinamide, mytatrienediol, ornithine, y-oryzanol, pantethine,pentaerythritol tetraacetate, alpha-phenylbutyramide, pirozadil,probucol (lorelco), p-sitosterol, sultosilic acid-piperazine salt,tiadenol, triparanol and xenbucin. In some embodiments,antihyperlipoproteinemic agents can further comprise anaryloxyalkanoicifibric acid derivative, a resin/bile acid sequesterant,an HMG CoA reductase inhibitor, a nicotinic acid derivative, a thyroidhormone or thyroid hormone analog, a miscellaneous agent or acombination thereof.

In another embodiment, administration of an agent that aids in theremoval or prevention of blood clots may be combined with administrationof a modulator, particularly in treatment of athersclerosis andvasculature (e.g., arterial) blockages. Examples of antithromboticand/or fibrinolytic agents can include but are not limited toanticoagulants, anticoagulant antagonists, antiplatelet agents,thrombolytic agents, throinbolytic agent antagonists or combinationsthereof. Antithrombotic agents that can be included are those that areadministered orally, such as, for example, aspirin and warfarin(coumadin).

Anticoagulants can include but are not limited to acenocoumarol, ancrod,anisindione, bromindione, clorindione, coumetarol, cyclocumarol, dextransulfate sodium, dicumarol, diphenadione, ethyl biscoumacetate,ethylidene dicoumarol, fluindione, heparin, hirudin, lyapolate sodiuim,oxazidione, pentosan polysulfate, phenindione, phenprocoumon, phosvitin,picotamide, tioclomarol and warfarin.

Antiplatelet agents can include but are not limited to aspirin, adextran, dipyridamole (persantin), heparin, sulfinpyranone (anturane)and ticlopidine (ticlid).

Thrombolytic agents can include but are not limited to tissueplasminogen activator (activase), plasmin, pro-urokinase, urokinase(abbokinase) streptokinase (streptase) and anistreplasel APSAC(eminase).

In one embodiment, the therapeutic intervention is an antiarrhythmicagent. Antiarrhythmic agents can include, but are not limited to Class Iantiarrhythmic agents (sodium channel blockers), Class II antiarrhythmicagents (beta-adrenergic blockers), Class III antiarrhythmic agents(repolarization prolonging drugs), Class IV antiarrhythmic agents(calcium channel blockers) and miscellaneous antiarrhythric agents.Examples of sodium channel blockers can include but are not limited toClass IA, Class IB and Class IC antiarrhythmic agents. Non-limitingexamples of Class IA antiarrhythmic agents include disppyramide(norpace), procainamide (pronestyl) and quinidine (quinidex). Examplesof Class IB antiarrhythmic agents can include but are not limited tolidocaine (xylocalne), tocamide (tonocard) and mexiletine (mexitil).Examples of Class IC antiarrhythmic agents can include but are notlimited to encamide (enkaid) and flecamide (tambocor).

Examples of a beta blocker, otherwise known as a p-adrenergic blocker, ap-adrenergic antagonist or a Class II antiarrhythmic agent, can includebut are not limited to acebutolol (sectral), alprenolol, amosulalol,arotinolol, atenolol, befunolol, betaxolol, bevantolol, bisoprolol,bopindolol, bucumolol, bufetolol, bufuralol, bunitrolol, bupranolol,butidrine hydrochloride, butofilolol, carazolol, carteolol, carvedilol,celiprolol, cetamolol, cloranolol, dilevalol, epanolol, esmolol(brevibloc), indenolol, labetalol, levobunolol, mepindolol,metipranolol, metoprolol, moprolol, nadolol, nadoxolol, nifenalol,nipradilol, oxprenolol, penbutolol, pindolol, practolol, pronethalol,propanolol (inderal), sotalol (betapace), sulfinalol, talinolol,tertatolol, timolol, toliprolol and xibinolol. In some embodiments, thebeta blocker can comprise an aryloxypropanolamine derivative. Examplesof aryloxypropanolamine derivatives can include but are not limited toacebutolol, alprenolol, arotinolol, atenolol, betaxolol, bevantolol,bisoprolol, bopindolol, bunitrolol, butofilolol, carazolol, carteolol,carvedilol, celiprolol, cetamolol, epanolol, indenolol, mepindolol,metipranolol, metoprolol, mrnoprolol, nadolol, nipradilol, oxprenolol,penbutolol, pindolol, propanolol, talinolol, tertatolol, tinolol andtoliprolol.

Examples of agents that prolong repolarization, also known as a ClassIII antiarrhythmic agent, can include but are not limited to includeamiodarone (cordarone) and sotalol (betapace).

Examples of a calcium channel blocker, otherwise known as a Class IVantiarrhythmic agent, can include but are not limited to anarylalkylamine (e.g., bepridile, diltiazem, fendiline, gallopamil,prenylamine, terodiline, verapamil), a dihydropyridine derivative(felodipine, isradipine, nicardipine, nifedipine, nimodipine,nisoldipine, nitrendipine) a piperazinde derivative (e.g., cinnarizine,flunarizine, lidoflazine) or a micellaneous calcium channel blocker suchas bencyclane, etafenone, magnesium, mibefradil or perhexyline. In someembodiments, a calcium channel blocker comprises a long-actingdihydropyridine (nifedipine-type) calcium antagonist.

Examples of antihypertensive agents can include but are not limited tosympatholytic, alpha/beta blockers, alpha-blockers, anti-angiotensin IIagents, beta blockers, calcium channel blockers, vasodilators andmiscellaneous antihypertensives.

Examples of an alpha blocker, also known as an α-adrenergic blocker oran α-adrenergic antagonist, can include but are not limited to,amosulalol, arotinolol, dapiprazole, doxazosin, ergoloid mesylates,fenspiride, indoramin, labetalol, nicergoline, prazosin, terazosin,tolazoline, trimazosin and yohimbine. In certain embodiments, analpha-blocker may comprise a quinazoline derivative. Quinazolinederivatives can include but are not limited to alfuzosin, bunazosin,doxazosin, prazosin, terazosin and trimazosin. The antihypertensiveagent may be both an alpha and beta-adrenergic antagonist. Examples ofan alpha/beta blocker can include but are not limited to labetalol(normodyne, trandate).

Examples of anti-angiotensin II agents can include but are not limitedto angiotensin converting enzyme inhibitors and angiotensin II receptorantagonists. Angiotensin converting enzyme inhibitors (ACE inhibitors)can include but are not limited to alacepril, enalapril (vasotec),captopril, cilazapril, delapril, enalaprilat, fosinopril, lisinopril,moveltopril, perindopril, quinapril and ramipril. Examples of anangiotensin II receptor blocker, also known as an angiotensin IIreceptor antagonist, an ANG receptor blocker or an ANG-II type-Ireceptor blocker (ARBS), include but are not limited toangiocandesartan, eprosartan, irbesartan, losartan and valsartan.

Examples of a sympatholytic include a centrally acting sympatholytic ora peripherially acting sympatholytic. Examples of a centrally actingsympatholytic, also known as a central nervous system (CNS)sympatholytic, can include but are not limited to clonidine (catapres),guanabenz (wytensin) guanfacine (tenex) and methyldopa (aldomet).

Examples of a peripherally acting sympatholytic can include but are notlimited to a ganglion blocking agent, an adrenergic neuron blockingagent, beta-adrenergic blocking agent or an alphal-adrenergic blockingagent. Examples of a ganglion blocking agent include mecarnylamine(inversine) and trimethaphan (arfonad). Examples of an adrenergic neuronblocking agent can include but are not limited to guanethidine (ismelin)and reserpine (serpasil).

Examples of a beta-adrenergic blocker can include but are not limited toacenitolol (sectral), atenolol (tenormin), betaxolol (kerlone),carteolol (cartrol), labetalol (normodyne, trandate), metoprolol(lopressor), nadanol (corgard), penbutolol (levatol), pindolol (visken),propranolol (inderal) and timolol (blocadren).

Examples of alphal-adrenergic blocker can include but are not limited toprazosin (minipress), doxazocin (cardura) and terazosin (hytrin).

The therapeutic intervention can also comprise a vasodilator (e.g., acerebral vasodilator, a coronary vasodilator or a peripheralvasodilator). In other embodiments, a vasodilator comprises a coronaryvasodilator. Examples of a coronary vasodilator include but are notlimited to amotriphene, bendazol, benfurodil hemisuccinate,benziodarone, chlioracizine, chromonar, clobenfurol, clonitrate,dilazep, dipyridamole, droprenilamine, efloxate, erythrityltetranitrane, etafenone, fendiline, floredil, ganglefene, herestrolbis(p-dinoeylaminoethyl ether), hexobendine, itramin tosylate, khellin,lidoflanine, mannitol hexanitrane, medibazine, nicorglycerin,pentaerythritol tetranitrate, pentrinitrol, perhexyline, pimefyiline,trapidil, tricromyl, trimeG6PDidine, trolnitrate phosphate andvisnadine. In some embodiments, a vasodilator can comprise a chronictherapy vasodilator or a hypertensive emergency vasodilator. Examples ofa chronic therapy vasodilator can include but are not limited tohydralazine (apresoline) and minoxidil (loniten). Examples of ahypertensive emergency vasodilator can include but are not limited tonitroprusside (nipride), diazoxide (hyperstat IV), hydralazine(apresoline), minoxidil (loniten) and verapamil.

Examples of antihypertensives can also include, but are not limited to,ajmaline, gamma-amino butyric acid, bufeniode, cicletainine,ciclosidomine, a cryptenamine tannate, fenoldopam, flosequinan,ketanserin, mebutamate, mecamylamine, methyldopa, methyl 4-pyridylketone thiosemicarbazone, muzo limine, pargyline, pempidine, pinacidil,piperoxan, primaperone, a protoveratrine, raubasine, rescimetol,rilmenidene, saralasin, sodium nitrorusside, ticrynafen, trimethaphancamsylate, tyrosinase and urapidil.

In certain embodiments, an antihypertensive can comprise anarylethanolamine derivative, a benzothiadiazine derivative, aN-carboxyalkyl(peptide/lactam) derivative, a dihydropyridine derivative,a guanidine derivative, a hydrazines/phthalazine, an imidazolederivative, a quaternary ammoniam compound, a reserpine derivative or asuflonamide derivative. Examples of arylethanolamine derivatives caninclude but are not limited to amosulalol, bufuralol, dilevalol,labetalol, pronethalol, sotalol and sulfinalol. Examples ofbenzothiadiazine derivatives can include but are not limited toalthizide, bendroflumethiazide, benzthiazide, benzylhydrochlorothiazide,buthiazide, chlorothiazide, chlorthalidone, cyclopenthiazide,cyclothiazide, diazoxide, epithiazide, ethiazide, fenquizone,hydrochlorothizide, hydroflumethizide, methyclothiazide, meticrane,metolazone, paraflutizide, polythizide, tetrachlormethiazide andtrichlormethiazide. Examples of N-carboxyalkyl(peptide/lactam)derivatives can include but are not limited to alacepril, captopril,cilazapril, delapril, enalapril, enalaprilat, fosinopril, lisinopril,moveltipril, perindopril, quinapril and ramipril. Examples ofdihydropyridine derivatives can include but are not limited toamlodipine, felodipine, isradipine, nicardipine, nifedipine,nilvadipine, nisoldipine and nitrendipine. Examples of guanidinederivatives can include but are not limited to bethanidine, debrisoquin,guanabenz, guanacline, guanadrel, guanazodine, guanethidine, guanfacine,guanochlor, guanoxabenz and guanoxan. Examples ofhydrazines/phthalazines can include but are not limited to budralazine,cadralazine, dihydralazine, endralazine, hydracarbazine, hydralazine,pheniprazine, pildralazine and todralazine. Examples of imidazolederivatives can include but are not limited to clonidine, lofexidine,phentolamine, tiamenidine and tolonidine. Examples of quaternaryammonium compounds can include but are not limited to azamethoniumbromide, chlorisondamine chloride, hexamethonium, pentacyniumbis(methylsulfate), pentamethoniumi bromide, pentolinium tartrate,phenactropiniutm chloride and trimethidinium methosulfate. Examples ofreserpine derivatives can include but are not limited to bietaserpine,deserpidine, rescinnamine, reserpine and syrosingopine. Examples ofsulfonamide derivatives can include but are not limited to ambuside,clopamide, furosemide, indapamide, quinethazone, trip amide andxipamide.

Examples of agents for the treatment of congestive heart failure caninclude but are not limited to anti-angiotensin II agents,afterload-preload reduction treatment, diuretics and inotropic agents.

Examples of a diuretic can include but are not limited to a thiazide orbenzothiadiazine derivative (e.g., althiazide, bendroflumethazide,benzthiazide, benzylhydrochiorchlorothiazide, buthiazide,chlorothiazide, chlorothiazide, chlorthalidone, cyclopenthiazide,epithiazide, ethiazide, ethiazide, fenquizone, hydrochlorothiazide,hydroflumethiazide, methyclothiazide, meticrane, metolazone,paraflutizide, polythizide, tetrachloromethiazide, trichlormethiazide),an organomercurial (e.g., chlormerodrin, meralluride, mercamnphamide,mercaptomerin sodium, mercumallylic acid, mercumatilin dodium, mercurouschloride, mersalyl), a pteridine (e.g., furtherene, triamterene),purines (e.g., acefylline, 7-morpholinomethyltheophylline, pamobrom,protheobromine, theobromine), steroids including aldosterone antagonists(e.g., canrenone, oleandrin, spironolactone), a sulfonamide derivative(e.g., aceG6PDolamide, ambuside, azosemide, bumetanide, buG6PDolamide,chloraminophenami de, clofenamide, clopamide, clorexolone,diphenylmethane-4,4′-disulfonamide, disulfamide, ethoxzolamide,furosemide, indapamide, mefruside, methazolamide, piretanide,quinethazone, torasemide, trip amide, xipamide), a uracil (e.g.,aminometradine, amisometradine), a potassium sparing antagonist (e.g.,amiloride, triamterene) or a miscellaneous diuretic such as aminozine,arbutin, chlorazanil, ethacrynic acid, etozolin, hydracarbazine,isosorbide, mannitol, metochalcone, muzo limine, perhexyline, ticmafenand urea.

Examples of a positive inotropic agent, also known as a cardiotonic, caninclude but are not limited to acefylline, an acetyldigitoxin,2-amino-4-picoline, aminone, benfurodil hemisuccinate, bucladesine,cerberosine, camphotamide, convallatoxin, cymarin, denopamine,deslanoside, digitalin, digitalis, digitoxin, digoxin, dobutamine,dopamine, dopexamine, enoximone, erythrophleine, fenalcomine, gitalin,gitoxin, glycocyamine, heptaminol, hydrastinine, ibopamine, alanatoside, metamivam, milrinone, nerifolin, oleandrin, ouabain,oxyfedrine, prenalterol, proscillaridine, resibufogenin, scillaren,scillarenin, strphanthin, sulmazole, theobromine and xamoterol. In someembodiments, an intropic agent is a cardiac glycoside, a beta-adrenergicagonist or a phosphodiesterase inhibitor. Examples of a cardiacglycoside can include but are not limited to digoxin (lanoxin) anddigitoxin (crystodigin). Examples of a .beta.-adrenergic agonist includebut are not limited to albuterol, bambuterol, bitolterol, carbuterol,clenbuterol, clorprenaline, denop amine, dioxethedrine, dobutamine(dobutrex), dopamine (intropin), dopexamine, ephedrine, etafedrine,ethylnorepinephrine, fenoterol, formoterol, hexoprenaline, ibopamine,isoetharine, isoproterenol, mabuterol, metaproterenol, methoxyphenamine,oxyfedrine, pirbuterol, procaterol, protokylol, reproterol, rimiterol,ritodrine, soterenol, terbutaline, tretoquinol, tulobuterol andxamoterol. Examples of a phosphodiesterase inhibitor can include but arenot limited to aminone (inocor).

Antianginal agents may comprise organonitrates, calcium channelblockers, beta blockers and combinations thereof. Examples oforganonitrates, also known as nitrovasodilators, can include but are notlimited to nitroglycerin (nitro-bid, nitrostat), isosorbide dinitrate(isordil, sorbitrate) and amyl nitrate (aspirol, vaporole).

Endothelin (ET) is a 21-amino acid peptide that has potent physiologicand pathophysiologic effects that appear to be involved in thedevelopment of heart failure. The effects of ET are mediated throughinteraction with two classes of cell surface receptors. Inhibiting theability of ET to stimulate cells involves the use of agents that blockthe interaction of ET with its receptors. Examples of endothelinreceptor antagonists (ERA) can include but are not limited to Bosentan,Enrasentan, Ambrisentan, Darusentan, Tezosentan, Atrasentan, Avosentan,Clazosentan, Edonentan, sitaxsentan, TBC 3711, BQ 123, and BQ 788.

Histone deacetylase inhibitors that appear to have beneficial effectsthe treatment of pulmonary hypertension. Examples of histone deacetylaseinhibitors can include but are not limited to valproic acid andsuberoylanilide hydroxamic acid.

Evidence of therapeutic efficacy may be specific to the cardiovasculardisease being treated and can include evidence well known in the art.For example, evidence of therapeutic efficacy can include but is notlimited to improvement or alleviation of one or more symptoms of cardiachypertrophy, heart failure, or myocardial infarction in the subject, orin the delay in the transition from cardiac hypertrophy to heartfailure. The one or more improved or alleviated symptoms can include,for example, increased exercise capacity, increased cardiac ejectionvolume, decreased left ventricular end diastolic pressure, decreasedpulmonary capillary wedge pressure, increased cardiac output, increasedcardiac index, lowered pulmonary artery pressures, decreased leftventricular end systolic and diastolic dimensions, decreased cardiacfibrosis, decreased collagen deposition in cardiac muscle, decreasedleft and right ventricular wall stress, decreased wall tension,increased quality of life, and decreased disease related morbidity ormortality. Further, therapeutic efficacy can also include generalimprovements in the overall health of the patient, such as but notlimited to enhancement of patient life quality, increase in predictedsurvival rate, decrease in depression or decrease in rate of recurrenceof the indication (Physicians' Desk Reference (2010).

Efficacy of a therapeutic intervention can also include evaluating ormonitoring for the improvement of one or more symptoms of cardiachypertrophy, heart failure, or myocardial infarction in the subject, orfor the delay in the transition from cardiac hypertrophy to heartfailure. The one or more improved symptoms may include, for example,increased exercise capacity, increased cardiac ejection volume,decreased left ventricular end diastolic pressure, decreased pulmonarycapillary wedge pressure, increased cardiac output, increased cardiacindex, lowered pulmonary artery pressures, decreased left ventricularend systolic and diastolic dimensions, decreased cardiac fibrosis,decreased collagen deposition in cardiac muscle, decreased left andright ventricular wall stress, decreased wall tension, increased qualityof life and decreased disease related morbidity or mortality. Themeasured levels of plasma miRNAs may serve as a surrogate marker forefficacy of the therapeutic intervention.

Definitions

So that the invention may be more readily understood, certain technicaland scientific terms are specifically defined below. Unless specificallydefined elsewhere in this document, all other technical and scientificterms used herein have the meaning commonly understood by one ofordinary skill in the art to which this invention belongs.

As used herein, including the appended claims, the singular forms ofwords such as “a,” “an,” and “the,” include their corresponding pluralreferences unless the context clearly dictates otherwise.

“Activation,” “stimulation,” and “treatment,” as it applies to cells orto receptors, may have the same meaning, e.g., activation, stimulation,or treatment of a cell or receptor with a ligand, unless indicatedotherwise by the context or explicitly. “Ligand” encompasses natural andsynthetic ligands, e.g., cytokines, cytokine variants, analogues,muteins, and binding compounds derived from antibodies. “Ligand” alsoencompasses small molecules, e.g., peptide mimetics of cytokines andpeptide mimetics of antibodies. “Activation” can refer to cellactivation as regulated by internal mechanisms as well as by external orenvironmental factors. “Response,” e.g., of a cell, tissue, organ, ororganism, encompasses a change in biochemical or physiological behavior,e.g., concentration, density, adhesion, or migration within a biologicalcompartment, rate of gene expression, or state of differentiation, wherethe change is correlated with activation, stimulation, or treatment, orwith internal mechanisms such as genetic programming.

“Activity” of a molecule may describe or refer to the binding of themolecule to a ligand or to a receptor, to catalytic activity; to theability to stimulate gene expression or cell signaling, differentiation,or maturation; to antigenic activity, to the modulation of activities ofother molecules, and the like. “Activity” of a molecule may also referto activity in modulating or maintaining cell-to-cell interactions,e.g., adhesion, or activity in maintaining a structure of a cell, e.g.,cell membranes or cytoskeleton. “Activity” can also mean specificactivity, e.g., [catalytic activity]/[mg protein], or [immunologicalactivity]/[mg protein], concentration in a biological compartment, orthe like. “Activity” may refer to modulation of components of the innateor the adaptive immune systems.

“Administration” and “treatment,” as it applies to an animal, human,experimental subject, cell, tissue, organ, or biological fluid, refersto contact of an exogenous pharmaceutical, therapeutic, diagnosticagent, or composition to the animal, human, subject, cell, tissue,organ, or biological fluid. “Administration” and “treatment” can refer,e.g., to therapeutic, pharmacokinetic, diagnostic, research, andexperimental methods. Treatment of a cell encompasses contact of areagent to the cell, as well as contact of a reagent to a fluid, wherethe fluid is in contact with the cell. “Administration” and “treatment”also means in vitro and ex vivo treatments, e.g., of a cell, by areagent, diagnostic, binding compound, or by another cell. The term“subject” includes any organism, preferably an animal, more preferably amammal (e.g., rat, mouse, dog, cat, rabbit) and most preferably a human,including a human patient.

“Treat” or “treating” refers to administering a therapeutic agent, suchas a composition containing any of the present G6PD inhibitors, orsimilar compositions described herein, internally or externally to asubject or patient having one or more disease symptoms, or beingsuspected of having a disease or being at elevated at risk of acquiringa disease, for which the agent has therapeutic activity. Typically, theagent is administered in an amount effective to alleviate one or moredisease symptoms in the treated subject or population, whether byinducing the regression of or inhibiting the progression of suchsymptom(s) by any clinically measurable degree. The amount of atherapeutic agent that is effective to alleviate any particular diseasesymptom (also referred to as the “therapeutically effective amount”) mayvary according to factors such as the disease state, age, and weight ofthe patient, and the ability of the drug to elicit a desired response inthe subject whether a disease symptom has been alleviated can beassessed by any clinical measurement typically used by physicians orother skilled healthcare providers to assess the severity or progressionstatus of that symptom. While an embodiment of the present invention(e.g., a treatment method or article of manufacture) may not beeffective in alleviating the target disease symptom(s) in every subject,it should alleviate the target disease symptom(s) in a statisticallysignificant number of subjects as determined by any statistical testknown in the art such as the Student's t-test, the chit-test, the U-testaccording to Mann and Whitney, the Kruskal-Wallis test (H-test),Jonckheere-Terpstra-test and the Wilcoxon-test.

“Treatment,” as it applies to a human, veterinary, or research subject,refers to therapeutic treatment, prophylactic or preventative measures,to research and diagnostic applications.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that not all progeny willhave precisely identical DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

With respect to cells, the term “isolated” refers to a cell that hasbeen isolated from its natural environment (e.g., from a tissue orsubject). The term “cell line” refers to a population of cells capableof continuous or prolonged growth and division in vitro. Often, celllines are clonal populations derived from a single progenitor cell. Itis further known in the art that spontaneous or induced changes canoccur in karyotype during storage or transfer of such clonalpopulations. Therefore, cells derived from the cell line referred to maynot be precisely identical to the ancestral cells or cultures, and thecell line referred to includes such variants. As used herein, the terms“recombinant cell” refers to a cell into which an exogenous DNA segment,such as DNA segment that leads to the transcription of abiologically-active polypeptide or production of a biologically activenucleic acid such as an RNA, has been introduced.

Pharmaceutical Compositions and Administration

To prepare pharmaceutical or sterile compositions of the compositions ofthe present invention, the present compound may be admixed with apharmaceutically acceptable carrier or excipient. See, e.g., Remington'sPharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, MackPublishing Company, Easton, Pa. (1984).

Formulations of therapeutic and diagnostic agents may be prepared bymixing with acceptable carriers, excipients, or stabilizers in the formof, e.g., lyophilized powders, slurries, amorphous solution or solid,aqueous solutions or suspensions (see, e.g., Hardman, et al. (2001)Goodman and Gilman's The Pharmacological Basis of Therapeutics,McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science andPractice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.;Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: ParenteralMedications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, etal. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, MarcelDekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety,Marcel Dekker, Inc., New York, N.Y.).

Toxicity and therapeutic efficacy of the therapeutic compositions,administered alone or in combination with another agent, can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index (LD₅₀/ED₅₀). In particular aspects,therapeutic compositions exhibiting high therapeutic indices aredesirable. The data obtained from these cell culture assays and animalstudies can be used in formulating a range of dosage for use in human.The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration.

In an embodiment of the invention, a composition of the invention isadministered to a subject in accordance with the Physicians' DeskReference 2003 (Thomson Healthcare; 57th edition (Nov. 1, 2002)).

The mode of administration can vary. Suitable routes of administrationinclude intranasal, nasal, oral, rectal, transmucosal, intestinal,parenteral; intramuscular, subcutaneous, intradermal, intramedullary,intrathecal, direct intraventricular, intravenous, intraperitoneal,intraocular, inhalation, insufflation, topical, cutaneous, transdermal,or intra-arterial.

In particular embodiments, the composition or therapeutic can beadministered by an invasive route such as by injection (see above). Infurther embodiments of the invention, the composition, therapeutic, orpharmaceutical composition thereof, is administered intravenously,subcutaneously, intramuscularly, intraarterially, intra-articularly(e.g. in arthritis joints), intratumorally, or by inhalation, aerosoldelivery. Administration by non-invasive routes (e.g., orally; forexample, in a pill, capsule or tablet) is also within the scope of thepresent invention.

Compositions can be administered with medical devices known in the art.For example, a pharmaceutical composition of the invention can beadministered by injection with a hypodermic needle, including, e.g., aprefilled syringe or autoinjector.

The pharmaceutical compositions of the invention may also beadministered with a needleless hypodermic injection device; such as thedevices disclosed in U.S. Pat. Nos. 6,620,135; 6,096,002; 5,399,163;5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.

Alternately, one may administer the present compound or other G6PDinhibitors, or related compound in a local rather than systemic manner,for example, via injection of directly into the desired target site,often in a depot or sustained release formulation. Furthermore, one mayadminister the composition in a targeted drug delivery system, forexample, in a liposome coated with a tissue-specific antibody,targeting, for example, the liver, and more specifically hepatocytes.The liposomes will be targeted to and taken up selectively by thedesired tissue. Also included in a targeted drug delivery system isnanoparticle specific nasal or cardiac delivery of the viral vectors,RNAi, shRNA or other G6PD inhibitors, or G6PD-based compound, alone orin combination with an Ihh RNAi construct or similar inhibitors. Asummary of various delivery methods and techniques of siRNAadministration in ongoing clinical trials is provided in Zuckerman andDavis 2015; Nature Rev. Drug Discovery, Vol. 14: 843-856, December 2015.

The present composition can also comprise a delivery vehicle, includingliposomes, for administration to a subject, carriers and diluents andtheir salts, and/or can be present in pharmaceutically acceptableformulations. For example, methods for the delivery of nucleic acidmolecules are described in Akhtar et al., 1992, Trends Cell Bio., 2,139; DELIVERY STRATEGIES FOR ANTISENSE OLIGONUCLEOTIDE THERAPEUTICS, ed.Akbtar, 1995, Maurer et al., 1999, Mol. Membr. Biol., 16, 129-140;Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137, 165-192; and Leeet al., 2000, ACS Symp. Ser., 752, 184-192. U.S. Pat. No. 6,395,713 andPCT Publication No. WO 94/02595 further describe the general methods fordelivery of nucleic acid molecules. These protocols can be utilized forthe delivery of virtually any nucleic acid molecule.

The present composition can be administered to a desired target by avariety of methods known to those of skill in the art, including, butnot restricted to, encapsulation in liposomes, by iontophoresis, or byincorporation into other vehicles, such as hydrogels, cyclodextrins,biodegradable nanocapsules, and bioadhesive microspheres, or byproteinaceous vectors (see PCT Publication No. WO 00/53722).Alternatively, the therapeutic/vehicle combination is locally deliveredby direct injection or by use of an infusion pump. Direct injection ofthe composition, whether subcutaneous, intramuscular, or intradermal,can take place using standard needle and syringe methodologies, or byneedle-free technologies such as those described in Conry et al., 1999,Clin. Cancer Res., 5, 2330-2337 and PCT Publication No. WO 99/3 1262.

Therapeutic compositions comprising surface-modified liposomescontaining poly(ethylene glycol) lipids (PEG-modified, orlong-circulating liposomes or stealth liposomes) may also be suitablyemployed in the methods of the invention. These formulations offer amethod for increasing the accumulation of drugs in target tissues. Thisclass of drug carriers resists opsonization and elimination by themononuclear phagocytic system (MPS or RES), thereby enabling longerblood circulation times and enhanced tissue exposure for theencapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwataet al., Chem. Pharm. Bull. 1995, 43, 1005-1011). The long-circulatingliposomes enhance the pharmacokinetics and pharmacodynamics of DNA andRNA, particularly compared to conventional cationic liposomes which areknown to accumulate in tissues of the MPS (Liu et al., J. Biol. Chem.1995, 42, 24864-24870; PCT Publication No. WO 96/10391; PCT PublicationNo. WO 96/10390; and PCT Publication No. WO 96/10392). Long-circulatingliposomes are also likely to protect drugs from nuclease degradation toa greater extent compared to cationic liposomes, based on their abilityto avoid accumulation in metabolically aggressive MPS tissues such asthe liver and spleen.

The administration regimen depends on several factors, including theserum or tissue turnover rate of the therapeutic composition, the levelof symptoms, and the accessibility of the target cells in the biologicalmatrix. Preferably, the administration regimen delivers sufficienttherapeutic composition to effect improvement in the target diseasestate, while simultaneously minimizing undesired side effects.Accordingly, the amount of biologic delivered depends in part on theparticular therapeutic composition and the severity of the conditionbeing treated.

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment. Generally, the dose begins with an amount somewhat less thanthe optimum dose and it is increased by small increments thereafteruntil the desired or optimum effect is achieved relative to any negativeside effects. Important diagnostic measures include those of symptomsof, e.g., the inflammation or level of inflammatory cytokines produced.In general, it is desirable that a biologic that will be used is derivedfrom the same species as the animal targeted for treatment, therebyminimizing any immune response to the reagent.

As used herein, “inhibit” or “treat” or “treatment” includes apostponement of development of the symptoms associated with a disorderand/or a reduction in the severity of the symptoms of such disorder. Theterms further include ameliorating existing uncontrolled or unwantedsymptoms, preventing additional symptoms, and ameliorating or preventingthe underlying causes of such symptoms. Thus, the terms denote that abeneficial result has been conferred on a vertebrate subject with adisorder, disease or symptom, or with the potential to develop such adisorder, disease or symptom.

As used herein, the terms “therapeutically effective amount”,“therapeutically effective dose” and “effective amount” refer to anamount of the present compound or other G6PD inhibitors or inhibitorcompound of the invention that, when administered alone or incombination with an additional therapeutic agent to a cell, tissue, orsubject, is effective to cause a measurable improvement in one or moresymptoms of a disease or condition or the progression of such disease orcondition. A therapeutically effective dose further refers to thatamount of the compound sufficient to result in at least partialamelioration of symptoms, e.g., treatment, healing, prevention oramelioration of the relevant medical condition, or an increase in rateof treatment, healing, prevention or amelioration of such conditions.When applied to an individual active ingredient administered alone, atherapeutically effective dose refers to that ingredient alone. Whenapplied to a combination, a therapeutically effective dose refers tocombined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously. An effective amount of a therapeutic will result in animprovement of a diagnostic measure or parameter by at least 10%;usually by at least 20%; preferably at least about 30%; more preferablyat least 40%, and most preferably by at least 50%. An effective amountcan also result in an improvement in a subjective measure in cases wheresubjective measures are used to assess disease severity.

Kits

The present invention also provides kits comprising the components ofthe combinations of the invention in kit form. A kit of the presentinvention includes one or more components including, but not limited to,one or more G6PD inhibitors as discussed herein, in association with oneor more additional components including, but not limited to apharmaceutically acceptable carrier and/or a chemotherapeutic agent, asdiscussed herein. The present compound, composition and/or thetherapeutic agent can be formulated as a pure composition or incombination with a pharmaceutically acceptable carrier, in apharmaceutical composition.

Kits may also include primers, buffers, and probes along withinstructions for determining elevated levels of nucleic acid, proteins,or protein fragments of G6PD, or any combination thereof.

In one embodiment, a kit includes the present compounds/composition ofthe invention or a pharmaceutical composition thereof in one container(e.g., in a sterile glass or plastic vial) and a pharmaceuticalcomposition thereof and/or a chemotherapeutic agent in another container(e.g., in a sterile glass or plastic vial).

In another embodiment of the invention, the kit comprises a combinationof the invention, including the present compound or other G6PDinhibitors, or G6PD-based inhibitor compounds, along with apharmaceutically acceptable carrier, optionally in combination with oneor more therapeutic agent components formulated together, optionally, ina pharmaceutical composition, in a single, common container.

If the kit includes a pharmaceutical composition for parenteraladministration to a subject, the kit can include a device for performingsuch administration. For example, the kit can include one or morehypodermic needles or other injection devices as discussed above.

The kit can include a package insert including information concerningthe pharmaceutical compositions and dosage forms in the kit. Generally,such information aids patients and physicians in using the enclosedpharmaceutical compositions and dosage forms effectively and safely. Forexample, the following information regarding a combination of theinvention may be supplied in the insert: pharmacokinetics,pharmacodynamics, clinical studies, efficacy parameters, indications andusage, contraindications, warnings, precautions, adverse reactions,overdosage, proper dosage and administration, how supplied, properstorage conditions, references, manufacturer/distributor information andpatent information.

General Methods

Standard methods in molecular biology are described Sambrook, Fritschand Maniatis (1982 & 1989 2^(nd) Edition, 2001 3^(rd) Edition) MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Sambrook and Russell (2001) Molecular Cloning,3^(rd) ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego,Calif.). Standard methods also appear in Ausbel, et al. (2001) CurrentProtocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. NewYork, N.Y., which describes cloning in bacterial cells and DNAmutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2),glycoconjugates and protein expression (Vol. 3), and bioinformatics(Vol. 4).

Methods for protein purification including immunoprecipitation,chromatography, electrophoresis, centrifugation, and crystallization aredescribed (Coligan, et al. (2000) Current Protocols in Protein Science,Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis,chemical modification, post-translational modification, production offusion proteins, glycosylation of proteins are described (see, e.g.,Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2,John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) CurrentProtocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY,NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for LifeScience Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech(2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production,purification, and fragmentation of polyclonal and monoclonal antibodiesare described (Coligan, et al. (2001) Current Protocols in Immunology,Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999)Using Antibodies, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Harlow and Lane, supra). Standard techniques forcharacterizing ligand/receptor interactions are available (see, e.g.,Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, JohnWiley, Inc., New York).

EXAMPLES Example 1: Androstane- and Preganane-Derivative as anEfficacious and Specific G6PD Inhibitors

G6PD deficiency is common in humans, and several point mutations havebeen found in this enzyme in different ethnic groups around the world.Epidemiological studies suggest that individuals who harbor aMediterranean-type non-synonymous mutation [single nucleotidepolymorphism in exon 6: dbSNP rs5030868] have 80% less G6PD activity ascompared to normal individuals and are less likely to havecardiovascular diseases (Gupte (2008): Curr Opin Investig Drugs;9:993-1000), including sickle cell anemia-associated PH.

Several compounds with approximately 10-fold improved potency in anenzyme assay were identified, and this improved activity translated toefficacy in a cellular assay. The steroid inhibition of G6PD has beensubstantially developed; the 3β-alcohol can be replaced with 3β-H-bonddonors such as sulfamide, sulfonamide, urea, and carbamate. Improvedpotency was achieved by replacing the androstane nucleus with a pregnanenucleus, provided a ketone at C-20 is present. For pregnan-20-onesincorporation of a 21-hydroxyl group is often beneficial. The novelcompounds generally have good physicochemical properties and in vitroDMPK parameters. From >40 derivatives prepared three derivatives(Formula VI, Formula VII, Formula VIII) had IC50 in 1-10 μM ranges toinhibit G6PD activity. Also, these drugs displayed >100-fold efficacy toinhibit G6PD activity in vitro and in vivo than dehydroepiandrosteroneand epiandrosterone. These drugs also showed good cardiovascularactivity over other derivatives. Any modifications in the body of thenucleus (shown with a circle) of these drugs dampened or eliminatedtheir cardiovascular actions. These potent steroid inhibitors withpotential therapeutic utility will be beneficial in inhibiting G6PD andameliorating cardiovascular diseases.

Example 2: Synthesis of G6PD InhibitorsN-Ethyl-N′-[(3β,5α)-17-oxoandrostan-3-yl]urea (Compound 7e)

To a stirred solution of (3β,5α)-3-aminoandrostan-17-one, cyclic1,2-ethanediyl acetal 6 (350 mg, 1.05 mmol) in DCM (5 mL) at 20° C.under nitrogen was added ethyl isocyanate (112 mg, 1.57 mmol). Themixture was stirred overnight, and then DCM and water were added beforethe mixture was passed through a hydrophobic frit. The DCM phase wasconcentrated to give the intermediate ketal as a white solid (472 mg).The 17-ketal intermediate (472 mg) was deprotected in a similar mannerto compound 7a (as described in Hamilton et al., Novel SteroidInhibitors of Glucose 6-Phosphate Dehydrogenase; J. Med. Chem. 2012,55:4431-4445) and the crude product chromatographed on silica (25 gSNAP, DCM/ethyl acetate gradient elution), affording the product 7e as awhite solid (293 mg, 77%). 1H NMR (CDCl3): δ 0.82 (s, 3H), 0.86 (s, 3H),1.14 (t, J=7.3 Hz, 3H), 2.38-2.50 (m, 1H), 3.20 (q, J=7.2 Hz, 2H),3.46-3.59 (m, 1H), 4.35 (br s, 1H). 1H NMR (DMSO-d6): δ 0.78 (s, 6H),0.96 (t, J=7.2 Hz, 3H), 2.32-2.44 (m, 1H), 2.92-3.03 (m, 2H), 3.22-3.37(m, 1H), 5.58-5.65 (m, 1H). 13C NMR (CDCl3): δ 12.3, 13.8, 15.5, 20.4,21.8, 28.3, 29.6, 30.8, 31.5, 35.1, 35.3, 35.6, 35.9, 36.2, 37.5, 45.5,47.8, 49.7, 51.4, 54.4, 157.6, 221.4. LC-MS m/z 361.2 [M+1]+, 100%purity; m/z 359.2 [M−1]−, 100% purity. HRMS (ESI) m/z [M+H]+m/zcalculated for C₂₂H₃₇N₂O₂: 361.2850. Found: 361.2851.

Synthesis of 7a ((3β,5α)-3-Aminoandrostan-17-one)

(3β,5α)-3-Aminoandrostan-17-one, cyclic 1,2-ethanediyl acetal 6 (100 mg,0.30 mmol) was dissolved in acetone (1 mL), DCM (1 mL) and THF (4 mL).Then 2 M HCl (0.5 mL, 1 mmol) was added and the mixture stirredovernight. The reaction mixture was basified with 1 M NaOH. DCM wasadded and the mixture stirred before passing through a hydrophobic frit.The organic phase was concentrated and chromatographed on silica (10 gSNAP cartridge, DCM/MeOH gradient elution) to give the product 7a as awhite solid (10 mg, 12%). ¹H NMR (CDCl₃): δ 0.84 (s, 3H), 0.88 (s, 3H),2.40-2.52 (m, 1H), 2.70-2.82 (m, 1H). ¹³C NMR (CDCl₃): δ 11.2, 12.7,19.3, 20.7, 27.3, 29.8, 30.4, 31.0, 34.0, 34.6, 34.7, 34.8, 36.4, 37.6,44.4, 46.7, 49.9, 50.4, 220.3. HRMS (ESI) m/z [M+H]⁺ calcd for C₁₉H₃₂NO:290.2479. Found: 290.2480 Hamilton et al., Novel Steroid Inhibitors ofGlucose 6-Phosphate Dehydrogenase; J. Med. Chem. 2012, 55:4431-4445.Hitchin et al., A novel scalable and stereospecific synthesis of 3α- and3β-amino-5α-androstan-17-ones and 3α- and 3β-amino-5α-pregnan-20-ones.Tetrahedron Lett., 2012, 53(23): 2868-2872.

N-[(3β,5α)-20-Oxopregnan-3-yl]methanesulfonamide (Compound 14f)

14f was prepared and purified in a similar manner to compound 7h, using(3β,5α)-3-aminopregnan-20-one, 20-cyclic 1,2-ethanediyl acetal 13 (390mg, 1.08 mmol) and methane sulfonyl chloride (0.13 mL, 1.62 mmol).Evaporation of the solvent afforded the product 14f as a white solid(255 mg, 60%). 1H NMR (CDCl3): δ 0.62 (s, 3H), 0.81 (s, 3H), 2.13 (s,3H), 2.50-2.58 (m, 1H), 3.00 (s, 3H), 3.25-3.38 (m, 1H). 13C NMR(CDCl3): δ 12.2, 13.5, 21.2, 22.8, 24.4, 28.4, 30.3, 31.6, 31.8, 35.3,35.4, 36.7, 37.4, 39.0, 42.3, 44.2, 45.5, 53.5, 54.1, 56.6, 63.8, 209.7.LC-MS m/z 394.1 [M−1]−, 100% purity. HRMS (ESI) m/z [M+Cl]− calcd forC₂₂H₃₇ClNO₃S: 430.2188. Found: 430.2201.

(3β,5α)-3,21-Dihydroxypregnan-20-one; Compound 34

To a stirred solution of (3β,5α)-21-bromo-3-hydroxypregnan-20-one 37(1192 mg, 3 mmol) in DMF (100 mL) and water (50 mL) was added sodiumhydroxide (144 mg, 3.6 mmol) and the mixture stirred for 30 min Themixture was then diluted with water and extracted with ethyl acetate.The combined extracts were washed with water, dried, and the solventevaporated. The residue was chromatographed on silica (isohexane/ethylacetate gradient elution) and recrystallized from diethyl etheraffording the product 34 as a white solid (761 mg, 76%); 1H NMR (CDCl3):δ 0.65 (s, 3H), 0.83 (s, 3H), 2.15-2.30 (m, 1H), 2.42-2.50 (m, 1H), 3.27(br s, 1H) 3.53-3.67 (m, 1H), 4.15, 4.23 (ABq, JAB=18.9 Hz, 2H); 1H NMR(DMSO-d6): δ 0.52 (s, 3H), 0.74 (s, 3H), 1.95-2.07 (m, 1H), 2.55 (m,1H), 4.02 (d, J=5.8 Hz, 2H), 4.42 (d, J=4.8 Hz, 1H), 4.89 (t, J=5.8 Hz,1H); 13C NMR (CDCl3): δ 12.3, 13.5, 21.2, 22.9, 24.5, 28.6, 31.5, 32.0,35.5, 37.0, 38.1, 38.8, 44.8, 45.0, 54.2, 56.8, 59.4, 69.4, 71.2, 77.2,210.4; HRMS (ESI) m/z [M+Cl]− Calcd for C21H34C103: 369.2201. Found:369.2211.

Alternative methods to synthesize the compounds are as follows.

(3β)-3,21-Dihydroxypregn-5-en-20-one. PD2124 (Compound 34)

(3β)-21-(acetyloxy)-3-hydroxy-pregn-5-en-20-one, (0.4000 g, 1.07 mmol)was mixed with ethanol (40 mL) under a nitrogen atmosphere. 10% Pd oncharcoal (25 mg) was added and the system degassed using nitrogen. Thereaction was degassed and placed under a hydrogen atmosphere. Themixture was stirred overnight at ambient temperature. Water was addedand the mixture passed through celite dampened with water. All volatileswere removed and the residue dissolved in diethyl ether, dried overmagnesium sulphate, filtered and the solvents removed to give a whitesolid. The crude material was loaded onto silica using dichloromethanethen purified using a 25 g Biotage SNAP cartridge—eluting with 0-100%Ethyl acetate/isohexane. Concentration of the clean fractions gave(3β)-21-(acetyloxy)-3-hydroxy-pregn-5-en-20-one (55 mg, 12%) as a whitepowder.

To a solution of (3β)-21-(acetyloxy)-3-hydroxy-pregn-5-en-20-one (500mg, 1.33 mmol) in methanol (10 mL) and water (1 mL) was added potassiumcarbonate (184.52 mg, 1.33 mmol) and the mixture stirred at reflux for30 min TLC (silica, EtOAc eluant) showed complete conversion of startingester (Rf 0.7) to a more polar product (Rf 0.6). The reaction mixturewas concentrated under reduced pressure, diluted with water andextracted with EtOAc. The combined extracts were washed with water,dried over anhydrous Na2SO₄ and concentrated under reduced pressure.This afforded a solid that was recrystallized from EtOAc affording(3β)-21-(acetyloxy)-3-hydroxypregn-5-en-20-one (164 mg).

To a solution of (3β)-21-(acetyloxy)-3-hydroxypregn-5-en-20-one (500 mg,1.34 mmol) in methanol (10 mL) and water (1 mL) was added potassiumcarbonate (185 mg, 1.34 mmol) and the mixture stirred at reflux for 30min. The reaction mixture was concentrated, diluted with water andextracted with ethyl acetate. The combined extracts were washed withwater, dried and the solvent evaporated. The solid was recrystallizedfrom ethyl acetate affording the product as a white solid (164 mg, 37%);¹H NMR (CDCl₃): δ 0.67 (s, 3H), 1.02 (s, 3H), 2.43-2.52 (m, 1H),3.48-3.60 (m, 1H), 4.17, 4.25 (ABq, JAB=19.0 Hz, 2H), 5.34-3.39 (m, 1H);¹³C NMR (CDCl₃): δ 13.3, 19.4, 21.0, 22.9, 24.6, 31.6, 31.8, 31.9, 36.5,37.2, 38.6, 42.2, 44.8, 49.9, 57.0, 59.2, 69.4, 71.7, 121.3, 140.8,210.3.

N-Ethyl-N′-[(3β,5α)-17-oxoandrostan-3-yl]urea. PD2958 (Compound 7e)

(3α,5α)-3-Hydroxyandrostan-17-one, cyclic 1,2-ethanediyl acetal (8.17 g,24.4 mmol), phthalimide (3.59 g, 24.4 mmol), and TPP (7.08 g, 25.6 mmol)were dissolved in THF (175 mL), and the solution was cooled to 5° C.using an ice bath. DIAD (5.3 mL, 26.8 mmol) was added slowly maintainingthe temperature below 5° C. A white precipitate formed during thereaction. The mixture was stirred and warmed to ambient temperatureovernight under a nitrogen atmosphere. All volatiles were evaporated andmethanol (100 mL) was added and the mixture stirred for 30 min. Theprecipitate was filtered off and washed with methanol. The solid wasdried under vacuum to give 2-[(3β,5α)-17-Oxoandrostan-3-yl, 17-cyclic1,2-ethanediylacetal]-1H-isoindole-1,3(2H)-dione as a white solid (7.67g, 68%). 1H NMR (CDCl3): δ 0.87 (s, 3H), 0.98 (s, 3H), 1.95-2.05 (m,1H), 2.22-2.49 (m, 2H), 3.85-3.97 (m, 4H), 4.13-4.24 (m, 1H), 7.68-7.74(m, 2H), 7.79-7.85 (m, 2H).

2-[(3β,5α)-17-Oxoandrostan-3-yl, 17-cyclic1,2-ethanediylacetal]-1H-isoindole-1,3(2H)-dione (7.67 g, 15.6 mmol) wasmixed with ethanol (100 mL) and hydrazine hydrate (11 mL, 226 mmol) andrefluxed overnight under a nitrogen atmosphere. The reaction mixture wascooled, and the solid was filtered off. The filtrate was evaporated todryness. The residue was partitioned between DCM and water, and thesolution passed through a hydrophobic frit. The DCM phase was evaporatedto dryness to give the (3β,5α)-3-aminoandrostan-17-one, cyclic1,2-ethanediyl acetal (5.34 g, 97%). 1H NMR (CDCl3): δ 0.79 (s, 3H),0.84 (s, 3H), 1.92-2.04 (m, 1H), 2.61-2.73 (m, 1H), 3.82-3.98 (m, 4H).

To a stirred solution of (3β,5α)-3-aminoandrostan-17-one, cyclic1,2-ethanediyl acetal (350 mg, 1.05 mmol) in DCM (5 mL) at 20° C. undernitrogen was added ethyl isocyanate (112 mg, 1.57 mmol). The mixture wasstirred overnight, and then DCM and water were added before the mixturewas passed through a hydrophobic frit. The DCM phase was concentrated togive the intermediate ketal as a white solid (472 mg). The 17-ketalintermediate (472 mg) was dissolved in acetone (1 mL), DCM (1 mL) andTHF (4 mL), 2 M HCl (0.5 mL, 1 mmol) was added and the mixture stirredovernight. The reaction mixture was basified with 1 M NaOH. DCM wasadded and the mixture stirred before passing through a hydrophobic frit.The organic phase was concentrated and chromatographed on silica (25 gSNAP, DCM/ethyl acetate gradient elution), affordingN-Ethyl-N′-[(3β,5α)-17-oxoandrostan-3-yl]urea as a white solid (293 mg,77%). ¹H NMR (CDCl₃): δ 0.82 (s, 3H), 0.86 (s, 3H), 1.14 (t, J=7.3 Hz,3H), 2.38-2.50 (m, 1H), 3.20 (q, J=7.2 Hz, 2H), 3.46-3.59 (m, 1H), 4.35(br s, 1H). 1H NMR (DMSO-d6): δ 0.78 (s, 6H), 0.96 (t, J=7.2 Hz, 3H),2.32-2.44 (m, 1H), 2.92-3.03 (m, 2H), 3.22-3.37 (m, 1H), 5.58-5.65 (m,1H). ¹³C NMR (CDCl3): δ 12.3, 13.8, 15.5, 20.4, 21.8, 28.3, 29.6, 30.8,31.5, 35.1, 35.3, 35.6, 35.9, 36.2, 37.5, 45.5, 47.8, 49.7, 51.4, 54.4,157.6, 221.4. LC-MS m/z 361.2 [M+H]+, 100% purity; m/z 359.2 [M−H]−,100% purity. HRMS (ESI) m/z+m/z calcd for C₂₂H₃₇N₂O₂: 361.2850. Found:361.2851.

N-[(3β,5α)-17-Oxoandrostan-3-yl]sulfamide. PD4091 (Compound 14f)

(3β,5α)-3-Hydroxyandrostan-17-one, cyclic 1,2-ethanediyl acetal (5.83 g,17.4 mmol), phthalimide (2.56 g, 17.4 mmol), and TPP (5.06 g, 18.3 mmol)were dissolved in THF (140 mL) and cooled to 5° C. using an ice bath.DIAD (3.8 mL, 19.2 mmol) was added slowly, maintaining the temperaturebelow 7° C. The yellow color was allowed to disappear between additions.The mixture was allowed to warm to ambient temperature overnight under anitrogen atmosphere. The solvent was evaporated and methanol was addedto the oil, forming a white precipitate. The suspension was stirred for30 min. The precipitate was filtered off and washed with methanol (100mL), affording 2-[(3β,5α)-17-Oxoandrostan-3-yl, 17-cyclic 1,2-ethanediylacetal]-1H-isoindole-1,3(2H)-dione as a white solid (5.93 g, 74%). ¹HNMR (CDCl₃): δ 0.77 (s, 3H), 0.78 (s, 3H), 3.75-3.86 (m, 4H), 4.41-4.42(m, 1H), 7.60-7.65 (m, 2H), 7.71-7.77 (m, 2H).

2-[(3β,5α)-17-Oxoandrostan-3-yl, 17-cyclic 1,2-ethanediylacetal]-1H-isoindole-1,3(2H)-dione (5.93 g, 12.8 mmol) was treated withethanol (180 mL) and hydrazine hydrate (8.0 mL, 164 mmol) and themixture heated at reflux overnight under a nitrogen atmosphere. Thereaction mixture was cooled and the solid precipitate was filtered off.The filtrate was evaporated and the residue partitioned between DCM andwater, and then passed through a hydrophobic frit. The DCM phase wasevaporated to give 2-[(3β,5α)-17-Oxoandrostan-3-yl, 17-cyclic1,2-ethanediyl acetal]-1H-isoindole-1,3(2H)-dione as a white solid (4.06g, 95%); ¹H NMR (CDCl₃): δ 0.dos (s, 3H), 0.76 (s, 3H), 1.85-1.94 (m,1H), 3.01-3.11 (br s, 1H), 3.75-3.87 (m, 4H).

A magnetically stirred solution of Sulfamide (481.26 mg, 5.01 mmol) and2-[(3β,5α)-17-Oxoandrostan-3-yl, 17-cyclic 1,2-ethanediylacetal]-1H-isoindole-1,3(2H)-dione (167. mg, 0.50 mmol) in 1,4-dioxane(5 mL) was heated at reflux with stirring for 48 h. The reaction mixturewas diluted with water and extracted with ethyl acetate. The combinedextracts were washed with saturated NaHCO₃ and water. The organic phasewas dried and evaporated to leave a residue which was treated with water(2 mL), MeOH (3 mL) and MeSO3H (0.4 mL) and stirred vigorously for 15min. The reaction mixture was basified with saturated NaHCO₃(˜10 mL) andpassed through a hydrophobic filter. The aqueous phase was washed withDCM. The organic phase was concentrated to dryness affording residuewhich was chromatographed over silica (isohexane/EtOAc eluant) to giveN-[(3β,5α)-17-Oxoandrostan-3-yl]sulfamide (115 mg, 51%) as an off-whitepowder. ¹H NMR (DMSO-d6): δ 0.77 (s, 3H), 0.78 (s, 3H), 2.32-2.44 (m,1H), 3.42-3.51 (m, 1H), 6.36 (br d, J=5.9 Hz, 1H), 6.40 (br s, 2H). ¹³CNMR (DMSO-d6): δ 11.4, 13.4, 19.6, 21.3, 26.2, 27.8, 30.5, 31.4, 31.9,33.1, 34.4, 35.2, 35.4, 38.7, 47.1, 48.2, 50.8, 53.6, 219.8. LC-MS m/z367.1 [M−1]−.

Experimental Methods

Flash chromatography was performed using pre-packed silica gelcartridges (KP-Sil SNAP, Biotage, Hengoed UK or RediSep Rf, Isco). Thinlayer chromatography was conducted with 5×10 cm plates coated with MerckType 60 F254 silica gel to a thickness of 0.25 mm. All reagents obtainedfrom commercial sources were used without further purification.Anhydrous solvents were obtained from the Sigma-Aldrich Chemical CompanyLtd. or Fisher Chemicals Ltd., and used without further drying. HPLCgrade solvents were obtained from Fisher Chemicals Ltd.

All compounds were >90% purity as determined by examination of both theLC-MS and 1H NMR spectra unless otherwise indicated. Where Cl or Br werepresent, expected isotopic distribution patterns were observed.

¹H NMR

Proton (¹H) and carbon (¹³C) NMR spectra were recorded on a 300 MHzBruker spectrometer. Solutions were typically prepared in eitherdeuterochloroform (CDCl₃) or deuterated dimethylsulfoxide (d6-DMSO) withchemical shifts referenced to tetramethylsilane (TMS) or deuteratedsolvent as an internal standard. ¹H NMR data are reported indicating thechemical shift (δ), the integration (e.g. 1H), the multiplicity (s,singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad;dd, doublet of doublets etc.) and the coupling constant (J) in Hz (appimplies apparent coupling on broadened signals). Deuterated solventswere obtained from the Sigma-Aldrich Chemical Company, Goss orFluorochem.

Analytical LC-MS.

LC-MS analyses were performed on a Waters Acquity UPLC system fittedwith BEH C18 1.7 μm columns (2.1×50 mm) and with UV diode arraydetection (210-400 nm). Positive and negative mass ion detection wasperformed using a Waters SQD detector. Analyses were performed witheither buffered acidic or basic solvents and gradients as detailedbelow:

Low pH:

Solvent A—Water+10 mM ammonium formate+0.1% formic acid

Solvent B—Acetonitrile+5% water+0.1% formic acid

High pH:

Solvent A—Water+10 mM ammonium hydrogen carbonate+0.1% ammonia solution

Solvent B—Acetonitrile+0.1% ammonia solution

Gradient:

Flow rate % Solvent Time (mL/min) A % Solvent B 0 0.6 95 5 1.2 0.6 5 951.7 0.6 5 95 1.8 0.6 95 5

Example 3

Mice were exposed to hypoxia (10% 02) or to ambient atmosphere (21% 02)for 5 weeks. These mice developed pulmonary hypertension (PH group).Mice were treated with PD2124 (Compound 34) s.c. for 1 week. Body weightand hematocrit was measured (FIG. 2).

Bovine pulmonary arteries were incubated in Krebs buffer with andwithout G6PD inhibitor PD2958 (2958) and PD2124 (2124) for 12 hr.Vascular smooth cell-specific protein myocardin (MYOCD) and smoothmuscle myosin heavy chain (MHY11) expression was increased by PD2958 (10microM) and PD2124 (1 microM) (FIGS. 3A and 3B).

Rat pulmonary artery smooth muscle cells were cultured in 15% DMEM mediafor 48 hr and cells were treated with newly synthesized G6PD drugs;PD109 (109), PD2124 (2124), PD2958 (2958) and PD4091 (4091) or vehiclecontrol (Con) for 72 hr. Cell number/proliferation and apoptosis wasdetermined by Cyquant and Caspase3/7 assay, respectively. Cell numberswere decreased by G6PD inhibitors. Cell apoptosis was increased byPD2958 (1 microM) N=5 in each group (FIGS. 4A and 4B).

Rat pulmonary artery smooth muscle cells were cultured in 15% DMEM mediafor 48 hr and cells were treated with newly synthesized G6PD drugs;PD2958 (2958) or vehicle control (Con) for 72 hr. Cells migration wasdetermined. Cell migration was abrogated by PD2958 (1 microM). N=5 ineach group (FIGS. 5A and 5B).

Bovine pulmonary artery rings were incubated in Krebs buffer andpre-contracted with KCl (30 mM). These pre-contracted rings relaxed byG6PD inhibitors; PD109 (109), PD2124 (2124), PD2958 (2958) and PD4091(4091) or dehydroepiandrosterone (DHEA) (FIG. 6).

Aortic rings of wild-type and G6PD-deficient pre-contracted with KCl (30mM). These rings from wild-type but not G6PD-deficient mice were relaxedin dose-dependent manner by PD2958 (2958) (FIG. 7).

Mice were divided in four groups; control (ctrl) exposed to ambientatmosphere (21% 02); pulmonary hypertension exposed to 10% 02 (PH); PHtreated with inactive G6PD inhibitor (PH_Placebo); and PH treated withactive G6PD inhibitor (PH_2124) for 5 wk. Placebo and 2124 were injectedS.C. (1.3 mg/Kg) for 1 wk from wk 4 to wk 5. G6PD inhibitor 2124 reducedand reversed pulmonary resistance determined as PAAT-to-ET ratio,arterial elastance, and TPR (FIGS. 8A-8C).

Mice were divided in four groups; control (ctrl) exposed to ambientatmosphere (21% O2); pulmonary hypertension exposed to 10% 02 (PH); PHtreated with inactive G6PD inhibitor (PH_Placebo); and PH treated withactive G6PD inhibitor (PH_2124) for 5 wk. Placebo and 2124 were injectedS.C. (1.3 mg/Kg) for 1 wk from wk 4 to wk 5. G6PD inhibitor 2124 reducedand reversed left ventricle (LV) stiffness and increased cardiac index(FIGS. 9A-9B).

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The invention is defined by the terms of theappended claims, along with the full scope of equivalents to which suchclaims are entitled. The specific embodiments described herein,including the following examples, are offered by way of example only,and do not by their details limit the scope of the invention.

All references cited herein are incorporated by reference to the sameextent as if each individual publication, database entry (e.g. Genbanksequences or GeneID entries), patent application, or patent, wasspecifically and individually indicated to be incorporated by reference.This statement of incorporation by reference is intended by Applicants,pursuant to 37 C.F.R. § 1.57(b)(1), to relate to each and everyindividual publication, database entry (e.g. Genbank sequences or GeneIDentries), patent application, or patent, each of which is clearlyidentified in compliance with 37 C.F.R. § 1.57(b)(2), even if suchcitation is not immediately adjacent to a dedicated statement ofincorporation by reference. The inclusion of dedicated statements ofincorporation by reference, if any, within the specification does not inany way weaken this general statement of incorporation by reference.Citation of the references herein is not intended as an admission thatthe reference is pertinent prior art, nor does it constitute anyadmission as to the contents or date of these publications or documents.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. Variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1-11. (canceled)
 12. A method for treating a cardiovascular disorderand/or a pulmonary disorder in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of acompound comprising N-((3β, 5α)-20-Oxopregnan-3-yl) methanesulfonamide,N-ethyl-N′-((3β, 5α)-17-oxoandrostan-3-yl)urea,(3β,5α)-3,21-Dihydroxypregnan-20-one, orN-((3β,5α)-17-Oxoandrostan-3-yl)sulfamide or any combination thereof, ora pharmaceutically acceptable salt (crystal and/or amorphous), non-saltamorphous form, solvate, poly-morph, tautomer or prodrug thereof,wherein the cardiovascular disorder and/or pulmonary disorder comprisescongestive heart failure, hypertrophic cardiomyopathy, diabeticcardiomyopathy, stroke, acute neointimal formation following iatrogenicinterventions, atherosclerosis, angiosarcoma, hemangiosarcoma, TimothySyndrome, or combinations thereof, and wherein the therapeuticallyeffective amount of the compound is between 10 to 300 mg/day.
 13. Themethod of claim 12, wherein the therapeutically effective amount of thecompound is between 30 to 200 mg/day.
 14. The method of claim 12,wherein the cardiovascular and/or pulmonary disorder comprisescongestive heart failure, hypertrophic cardiomyopathy, diabeticcardiomyopathy, stroke, acute neointimal formation following iatrogenicinterventions, atherosclerosis, or combinations thereof.
 15. The methodof claim 12, wherein the cardiovascular disorder and/or pulmonarydisorder comprises angiosarcoma, hemangioscarcoma, Timothy Syndrome, orcombinations thereof.
 16. The method of claim 12, further comprisingtreating the subject with a diuretic, a vasodilator, an inotropic agent,an angiotensin converting enzyme (ACE) inhibitor, a beta blocker, aneurohumoral blocker, an aldosterone antagonist, a histone deacetylaseinhibitor, erythropoietin, or combinations thereof.
 17. The method ofclaim 12, further comprising treating the subject with a histonedeacetylase inhibitor.